Human cytotoxic t-lymphocyte epitope and its agonist epitope from the non-variable number of tandem repeat sequence of muc-1

ABSTRACT

Novel MUC-1 epitopes outside the VNTR region are identified. In addition, the first agonist epitope of MUC-1 is described. The employment of agonist epitopes in peptide, protein and vector-based vaccine may well aid in the development of effective vaccines for a range of human cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 13/176,341, filed Jul. 5, 2011, which is adivisional of U.S. patent application Ser. No. 10/582,702, filed Apr.23, 2007, now U.S. Pat. No. 7,999,071, which claims the benefit of U.S.Provisional Application No. 60/529,329, filed Dec. 12, 2003, which isincorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 14,640 bytes ASCII (Text) file named“713778_ST25.txt,” created on Jun. 27, 2013.

FIELD OF THE INVENTION

CTL epitope sequences outside traditional MUC-1 immunogenic tumorantigens and HLA-anchor residues have been identified. In particular,the invention describes a method for T-cell activation by modifyingHLA-anchor residues to provide a stronger immune response to nativeantigens associated with solid tumors, leukemias, or lymphomas.

BACKGROUND OF THE INVENTION

The tumor-associated antigen MUC-1, or DF-3/MUC-1, is overexpressed onthe cell surface of many human adenocarcinomas such as ovarian, breast,pancreas, colorectal and prostate carcinoma, and hematologicalmalignancies including multiple myeloma and some B-cell non-Hodgkinlymphomas. While MUC-1 is expressed on some normal epithelial tissue onlumenal surfaces, it has been demonstrated that the apical localizationof MUC-1 is lost in tumor tissues. In addition, MUC-1 isunder-glycosylated in human adenocarcinomas as compared with normaltissues and thus the antigenic epitopes of the protein core are moreexposed. A high level of MUC-1 expression and secretion has also beenshown to be associated with poor prognosis and high metastaticpotential. It was initially demonstrated that histocompatibility complex(MHC)-unrestricted cytotoxic T cells could be established from subjectswith pancreatic carcinoma, ovarian cancer and multiple myeloma; these Tcells were shown to recognize the MUC-1 protein core in the 20 aminoacid variable number of tandem repeat (VNTR)² region. While the VNTRregion is immunogenic for MHC non-restricted CTL as well as for theproduction of MUC-1 specific antibodies, relatively limited informationis available with respect to the immunogenicity of the region outsidethe VNTR.

Current treatment of cancers include radiation therapy and chemotherapy,which have particularly adverse effects on a subject undergoing suchtherapies.

Accordingly, there is a need for improved, safer treatments that havelong-lasting protective effects for the prevention and treatment oftumors. In particular, there is a need for treatments that are morespecific and less toxic than the currently available therapeutic agents.

SUMMARY OF THE INVENTION

The invention describes the identification and characterization ofanti-tumor cytotoxic T lymphocyte (CTL) epitopes. In particular, MUC-1CTL epitopes in the non-variable number of tandem repeat (VNTR) regionextracellular region of MUC-1 are described. The VNTR is not a region ofMUC-1 which is traditionally known to have immunogenic epitopes. Theinvention also describes the generation of enhancer agonist epitopeswhich generate stronger immune cell reaction than native peptides.

In a preferred embodiment, the invention provides an isolated nucleicacid molecule which encodes an agonist polypeptide antigen derived froma tumor antigen, such as for example, MUC-1, wherein the agonistpolypeptide stimulates a stronger immune response as compared to anative polypeptide.

In another preferred embodiment, the agonist polypeptide binds to HLAmolecules with a high avidity as compared to native polypeptides.Preferably, the agonist polypeptide has a higher association constant(K_(a)) for HLA molecules than a native polypeptide. Also preferably,the agonist polypeptide has a lower dissociation constant (K_(d)) forHLA molecules than a native polypeptide.

In another preferred embodiment, the nucleic acid molecule encodes anagonist polypeptide up to about 12 amino acids in length. Preferably,the agonist polypeptide is derived from a mucin tumor antigen.

In another preferred embodiment, the agonist polypeptide is derived froma non-variable number of tandem repeats region of MUC-1. Preferably, theagonist polypeptide generates an immune response.

In one aspect of the invention, the generated immune response is acellular immune response. Cellular immune responses include cytotoxic Tcell responses, T helper cell responses, and B cell immune responses.

In another preferred embodiment, the invention provides a nucleic acidmolecule comprising a nucleic acid sequence corresponding to (i.e. thatcan code for) any one of the amino acid sequences as identified by SEQID NO: 1 through 19, fragments or variants thereof. SEQ ID NO: 1 through19 are identified by:

SEQ ID SEQ ID NO Peptide NO (peptide) sequence Nucleotide sequence(n.t.) 1 ATWGQDVTSV GCC/ACC/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 20 2ALWGQDVTSV GCC/CTG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 21 3 ALLVLVCVLVGCC/CTG/CTG/GTC/CTG/GTC/TGC/GTC/CTG/GTC 22 4 TISDVSVSDVACC/ATC/TCG/GAT/GTC/TCG/GTC/TCG/GAT/GTC 23 5 ALAIVYLIALGCC/CTG/GCC/ATC/GTC/TAC/CTG/ATC/GCC/CTG 24 6 VLVALAIVYLGTC/CTG/GTC/GCC/CTG/GCC/ATC/GTC/TAC/CTG 25 7 YLIALAVCQCTAC/CTG/ATC/GCC/CTG/GCC/GTC/TGC/CAA/TGC 26 8 WGQDVTSVPVTGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC/CCA/GTC 27 9 REGTINVHDVAGA/GAA/GGT/ACC/ATC/AAC/GTC/CAC/GAT/GTC 28 10 GTQSPFFLLLGGC/ACC/CAG/TCT/CCT/TTC/TTC/CTG/CTG/CTG 29 11 LAFREGTINVCTG/GCC/TTC/AGA/GAA/GGT/ACC/ATC/AAC/GTC 30 12 TLASHSTKTDACT/CTG/GCC/TCG/CAC/TCG/ACC/AAG/ACC/GAT 31 13 LQRDISEMFLCTG/CAA/AGA/GAT/ATC/TCG/GAA/ATG/TTC/CTG 32 14 AIWGQDVTSVGCC/ACT/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 33 15 ALWGQDVTSLGCC/CTG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/CTG 34 16 AMWGQDVTSVGCC/ATG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 35 17 AMWGQDVTSLGCC/ATG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/CTG 36 18 AIWGQDVTSLGCC/ACT/TGG/GGA/CAG/GAT/GTC/ACC/TCG/CTG 37 19 ALWGQDVTSV

In another preferred embodiment, the invention provides for a vectorcomprising an isolated nucleic acid molecule expressing any one of aminoacids identified by SEQ ID NO: 1 through 19.

In another preferred embodiment, the vector comprises nucleic acidmolecules encoding immune cell co-stimulatory molecules, such as forexample, B7-1, ICAM-1 and LFA-3.

In yet another preferred embodiment, the invention provides for thetransduction of dendritic cells with a vector comprising any one of themolecules as identified by SEQ ID NO: 1 through 19, fragments orvariants thereof, and optionally, immune cell co-stimulatory molecules,such as for example, B7-1, ICAM-1 and LFA-3.

In one aspect of the invention, dendritic cells transduced with thevector comprising any one of the molecules as identified by SEQ ID NO: 1through 19, fragments or variants thereof, and optionally, immune cellco-stimulatory molecules, generates an immune response, such asactivation of a cytotoxic T cell response.

In another preferred embodiment, the invention provides a nucleic acidvector comprising one or more nucleic acid sequences encodingpolypeptides as identified by any one of SEQ ID NO: 1 through 19,fragments or variants thereof, operably linked to an inducible promoter.

In another preferred embodiment the nucleic acid vector is a viralvector, plasmid and the like. Preferably the nucleic acid vectorcomprises an inducible promoter which is tissue specific, andoptionally, immune cell co-stimulatory molecules.

In another preferred embodiment, the vector comprising a nucleic acidsequence encoding any one of the polypeptides identified by SEQ ID NO: 1through 19.

In another preferred embodiment, the vector codes for any one of thepolypeptides identified by any one of SEQ ID NO: 1 through 19 having asequence identity to any one one of SEQ ID NO: 1 through 19 of at leastabout 10%, more preferably, 25%, even more preferably a sequenceidentity of about 40%, 50%, 60%, 70%, 80%, 90%, or 99.9% to any of theSEQ ID NO: 1 through 19.

In another preferred embodiment, the vector contains a sequenceidentified by any one of SEQ ID NO: 20 through 37 having a sequenceidentity to anyone one of SEQ ID NO: 20 through 37 of at least about10%, more preferably, 25%, even more preferably about 40%, 50%, 60%,70%, 80%, 90%, or 99.9% sequence identity to any one of SEQ ID NO:30-37.

In another preferred embodiment, the invention provides a host cellexpressing the polypeptide products of the vector as identified by anyone of SEQ ID NO: 1 through 19 having a sequence identity to anyone oneof SEQ ID NO: 1 through 19 of at least about 10%, more preferably, 25%,even more preferably about 40%, 50%, 60%, 70%, 80%, 90%, or 99.9%.Preferably the host cell is an antigen presenting cell, such as forexample, a monocyte/macrophage, dendritic cell or the like.

In another preferred embodiment, the invention provides a method fortreating a subject suffering from or susceptible to a MUC-1 tumorcomprising administering to a subject any one of the peptides identifiedby SEQ ID NO: 1 through 19, fragments or variants thereof.

In another preferred embodiment, the invention provides a method fortreating a subject suffering from or susceptible to a MUC-1 tumorcomprising administering to a subject any one of the nucleic acidsidentified by SEQ ID NO: 20 through 37, fragments or variants thereof.

In another preferred embodiment, the invention provides a method forgenerating an immune response to a MUC-1 tumor antigen comprisingadministering an isolated nucleic acid molecule in a therapeuticallyeffective dose sufficient to generate a cellular immune response,wherein the isolated nucleic acid molecule encodes any one ofpolypeptides identified by SEQ ID NO: 1 through 19, fragments orvariants thereof, and optionally immune cell co-stimulatory molecules.Preferably, the vector can express polypeptides as identified by any oneof SEQ ID NO: 1 through 19 having a sequence identity to anyone one ofSEQ ID NO: 1 through 19 of at least about 10%, more preferably, 25%,even more preferably about 40%, 50%, 60%, 70%, 80%, 90%, or 99.9%.

In another preferred embodiment, the invention provides for a method fortreating a subject suffering from or susceptible to a MUC-1 tumorcomprising isolating dendritic cells from a subject suffering fromcancer; and, treating the dendritic cells with one or more of thepolypeptides identified by SEQ ID NO: 1 through 19; fragments, andvariants thereof. Preferably, the treated dendritic cells areadministered to the subject.

In another preferred embodiment, the invention provides a method forgenerating an immune response to a weakly immunogenic antigen comprisingadministering to an subject a polypeptide with a high avidity for HLAfused to the weak immunogen.

In one aspect of the invention, the polypeptide comprises the HLAbinding fragment of SEQ ID NO: 19.

In another aspect of the invention, the weak immunogen is adifferentiation antigen, or a tumor antigen.

In another preferred embodiment, the HLA binding fragment of SEQ ID NO:19 is fused to a carcinoembryonic antigen, tumor antigen, self antigen,viral antigen and the like.

In another preferred embodiment, the invention provides for an isolatedpolypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1through 19, fragments or variants thereof.

In another preferred embodiment, the invention provides for apolypeptide identified by any one of SEQ ID NO: 1 through 19 having asequence identity to anyone one of SEQ ID NO: 1 through 19 of at leastabout 10%, more preferably, 25%, even more preferably about 40%, 50%,60%, 70%, 80%, 90%, or 99.9%.

In one aspect of the invention, the polypeptide comprises SEQ ID NO: 19.Preferably, the polypeptide binds to HLA molecules with a high avidityand has a higher association constant (K_(a)) for the HLA than a nativepolypeptide and/or a lower dissociation constant (K_(d)) for the HLAthan a native polypeptide.

In another aspect of the invention, the polypeptide is derived from amucin tumor antigen, preferably, the polypeptide is derived from anon-variable number of tandem repeats region of MUC-1.

In another aspect of the invention, antigen presentation, by antigenpresenting cells of the polypeptides induces an immune response,preferably a cellular immune response. For example, the cellular immuneresponse is a cytotoxic T cell response, a T helper cell response, or aB cell immune response.

In another preferred embodiment, the invention provides for an agonistpolypeptide comprising an amino acid sequence which is at least about60% identical to the amino acid sequence of SEQ ID NO: 1 through 19,fragments, or variants thereof, more preferably, the agonist polypeptidecomprises an amino acid sequence which is at least about 80% identicalto the amino acid sequence of SEQ ID NO: 1 through 19, more preferably,the agonist polypeptide comprises an amino acid sequence which is atleast about 90%, 95%, or 99.9% identical to the amino acid sequence ofSEQ ID NO: 1 through 19.

In another preferred embodiment, a method of treating a subjectsuffering from or susceptible to a MUC-1 tumor is disclosed. The methodmay include the isolating dendritic cells from a subject suffering fromcancer, treating the dendritic cells with one or more of polypeptidesidentified by SEQ ID NO: 1 through 19, activating peripheral bloodmononuclear cells with the treated dendritic cells, and administeringthe activated PBMC cells to the subject.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are graphs showing binding of MUC-1 peptide P-92 and theagonists P-93L and P-93I to HLA-A2 molecule. FIG. 1A is a graph showinguse of peptides at concentrations of 0-50 μg/ml. FIG. 1B is a graphshowing use of peptides at concentrations of 0-12.5 μg/ml. P-92 MUC-1peptide (open square), P-93L (closed square), P-93I (closed triangle).Results are expressed in mean fluorescence intensity (MFI).

FIG. 2 is a graph showing the comparison of the stability of the complexof the P-92, P-93L or P-93I peptide with HLA-A2. T2 cells were incubatedovernight with P-92 (open square), P-93L (closed square) or P-93I(closed triangle) peptide at a concentration of 50 μg/ml and then washedfree of unbound peptide and incubated with brefeldin-A to block deliveryof new class 1 molecules to the cell surface. At the indicated times,cells were stained for the presence of surface peptide-HLA-A2 complexes.Results are expressed in relative percentage of binding compared with100% at time 0.

FIG. 3 is a graph showing the ability of autologous B cells pulsed withnative MUC-1 peptide P-92 (open square), P-93L peptide (closed square)and P-93I peptide (closed triangle) to induce IFN-γ production byMUC-1-specific T cells. Peptides were used at concentrations of 0-6.25μg/ml. Results are expressed in pg/ml.

FIG. 4 is a graph showing the cytotoxicity of the MUC-1-specific T-celllines against C1R-A2 cells pulsed with P-92 and P-93L peptide.T-1191-P-93L against C1R-A2 pulsed with P-93L peptide (closed square),T-1191-P-93L against C1R-A2 pulsed with P-92 peptide (open square),T-1191-P-93L against C1R-A2 pulsed with CAP1-6D peptide (closed circle),T-1191-P-92 against C1R-A2 pulsed with P-93L peptide (closed triangle),T-1191-P-92 against C1R-A2 pulsed with P-92 peptide (open triangle),T-1191-P-92 against C1R-A2 pulsed with CAP1-6D peptide (open circle).E:T ratio=25:1 and 12.5:1 in a 16-h ¹¹¹In release assay. Bars=SD.

FIG. 5 shows a schematic representation of certain viral constructs.

FIG. 6 is a graphical representation of a flow cytometric analysis ofsurface marker expression on human DCs uninfected, infected with controlvector (V-WT), or infected with rV-CEA/MUC/TRICOM. DCs (1×10⁶) wereincubated in 1 ml of Opti-MEM medium at 37° C. with rV-CEA/MUC/TRICOM orcontrol vector (V-WT) for 1 hour, at an MOI of 5:1. The infected DCswere suspended in 10 ml of fresh, warm complete medium containing 100ng/ml of rhGM-CSF and 20 ng/ml of rhIL-4, and then cultured for 24hours. Numbers in each histogram indicate the percentage of positivecells and the mean fluorescence intensity (in parentheses).

FIG. 7 is a graphical representation of a flow cytometric analysis ofsurface marker expression on human DCs uninfected, infected with controlvector (FP-WT), or infected with rF-CEA/MUC/TRICOM. DCs (1×10⁶) wereincubated in 1 ml of Opti-MEM medium at 37° C. with rF-CEA/MUC/TRICOM orcontrol vector (FP-WT) for 2 hours, at an MOI of 40:1. The infected DCswere suspended in 10 ml of fresh, warm complete medium containing 100ng/ml of rhGM-CSF and 20 ng/ml of rhIL-4, and then cultured for 24 h.Numbers in each histogram indicate the percentage of positive cells andthe mean fluorescence intensity (in parentheses).

FIG. 8 shows an immunoblotting analysis of human DCs uninfected orinfected with rF-CEATRICOM, rF-MUC-1-TRICOM, rF-CEA/MUC/TRICOM andrV-CEA/MUC/TRICOM. Monoclonal antibodies COL-1 and DF-3 were used forthe detection of CEA and MUC-1, respectively.

FIG. 9 is the nucleotide sequence of the wMUC-1(6) vector (SEQ ID NO:41).

FIG. 10 is the amino acid sequence of wMUC-1(6) vector (SEQ ID NO: 42).

DETAILED DESCRIPTION OF THE INVENTION

We describe herein, inter alia, the identification of a novel class IHLA-A2 epitopes of MUC-1 that reside outside of the variable number oftandem repeat (VNTR) region that are important for immune basedtherapies in the treatment of cancer. We have demonstrated the abilityof these epitopes to activate human T cells as measured by IFN-γproduction. In particular, one epitope, ATWGQDVTSV (SEQ ID NO: 1), atamino acid position 92-101 and designated P-92), demonstrated thehighest level of binding the HLA-A2 and which induced the highest levelof IFN-γ in human T cells.

The invention also provides for the generation of enhancer agonistepitopes, as identified by epitope, ALWGQDVTSV, (SEQ ID NO: 19;designated P-93L).

Virtually all tumors express multiple tumor-associated antigens and thevast majority of them are heterogeneously expressed in tumor masses.This has been shown to be attributable to inherent antigenicheterogeneity, environmental factors in the tumor milieu such as spatialconfiguration, or antigenic drift due to therapeutic intervention. Thus,vaccines expressing multiple transgenes may well help to alleviate thisobstacle of antigenic heterogeneity. CEA is expressed on the vastmajority of colorectal, pancreatic, and gastric tumors, and inapproximately 70% of non-small-cell lung cancers, 50% of breast cancers,as well as other tumor types such as head and neck carcinoma and subsetsof ovarian carcinoma (Thompson J A, Grunert F, Zimmermann, W.Carcinoembryonic antigen gene family: molecular biology and clinicalperspectives. J Clin Lab Anal 1991; 5:344-66; and Robbins P F,Eggensperger D, Qi C F, Schlom J. Definition of the expression of thehuman carcinoembryonic antigen and non-specific cross-reacting antigenin human breast and lung carcinomas. Int J Cancer 1993; 53:892-7).MUC-1, on the other hand, is overexpressed on the vast majority ofcolorectal, pancreatic, breast and ovarian cancers as well as othercarcinoma types (Kufe D, Inghirami G, Abe M. Differential reactivity ofa novel monoclonal antibody (DF3) with human malignant versus benignbreast tumors. Hybridoma 1984; 3:223-32; Burchell J, Gendler S,Taylor-Papadimitriou J, et al. Development and characterization ofbreast cancer reactive monoclonal antibodies directed to the coreprotein of the human milk mucin. Cancer Res 1987; 47: 5476-82; Zotter S,Hageman P C, Lossnitzer A, Mooi W J, Hilgers J. Tissue and tumordistribution of human polymorphic epithelial mucin. Cancer Rev 1988;11-12: 55-101; Kotera Y, Fontenot J D, Pcher G, Metzgar R S, Finn O J.Humoral immunity against a tandem repeat epitope of human mucin MUC-1 insera from breast, pancreatic, and colon cancer patients. Cancer Res1994; 54:2856-60; and Goydos J S, Eler E, Whiteside T L, Finn O J, LotzeM T. A phase I trial of a synthetic mucin peptide vaccine. Induction ofspecific immune reactivity in patients with adenocarcinoma. J Surg Res1996; 63:298-304). Thus, either individual or multi-targeting of thesetwo antigens may prove advantageous for those cancer types expressingboth antigens.

In certain embodiments the nucleic acid molecule does not have asequence as described in FIG. 9.

In certain embodiments the peptide molecule does not have a sequence asdescribed in FIG. 10.

In certain embodiments the nucleic acid molecule has a sequence asdescribed in FIG. 9.

In certain embodiments the peptide molecule has a sequence as describedin FIG. 10.

In certain embodiments the nucleic acid molecule does not have about a30 nucleotide portion of consecutive nucleotides of a sequence asdescribed in FIG. 9.

In certain embodiments the peptide molecule does not have about a 30amino acid portion of consecutive amino acids of a sequence a sequenceas described in FIG. 10.

In certain embodiments the nucleic acid molecule has a sequence asdescribed in FIG. 9.

In certain embodiments the peptide molecule has a sequence as describedin FIG. 10.

In certain embodiments the nucleic acid molecule does not have about a30 nucleotide portion of consecutive nucleotides of a sequence asequence as described in FIGS. 7 and/or 8 of PCT Applications:PCT/US04/37810, filed Nov. 12, 2004 and PCT/US04/38643, filed Nov. 12,2004.

In certain embodiments the nucleic acid molecule has a sequence asdescribed in FIGS. 7 and/or 8 of PCT Application: PCT/US04/37810, filedNov. 12, 2004 and PCT/US04/38643, filed Nov. 12, 2004.

In certain embodiments the nucleic acid molecule does not have about a30 nucleotide portion of consecutive nucleotides of a sequence asdescribed in PCT/US04/37810, filed Nov. 12, 2004 or PCT/US04/38643,filed Nov. 12, 2004.

In certain embodiments the nucleic acid molecule has a sequence asdescribed in PCT/US04/37810, filed Nov. 12, 2004 or PCT/US04/38643,filed Nov. 12, 2004.

The following definitions of certain terms that are used herewith, areset forth below.

As used herein, “molecule” is used generically to encompass any vector,antibody, protein, drug and the like which are used in therapy and canbe detected in a subject by the methods of the invention. For example,multiple different types of nucleic acid delivery vectors encodingdifferent types of genes which may act together to promote a therapeuticeffect, or to increase the efficacy or selectivity of gene transferand/or gene expression in a cell. The nucleic acid delivery vector maybe provided as naked nucleic acids or in a delivery vehicle associatedwith one or more molecules for facilitating entry of a nucleic acid intoa cell. Suitable delivery vehicles include, but are not limited to:liposomal formulations, polypeptides; polysaccharides;lipopolysaccharides, viral formulations (e.g., including viruses, viralparticles, artificial viral envelopes and the like), cell deliveryvehicles, and the like.

As used herein, the term “administering a molecule to a cell” (e.g., anexpression vector, nucleic acid, cytokines, a delivery vehicle, agent,and the like) refers to transducing, transfecting, microinjecting,electroporating, or shooting, the cell with the molecule. In someaspects, molecules are introduced into a target cell by contacting thetarget cell with a delivery cell (e.g., by cell fusion or by lysing thedelivery cell when it is in proximity to the target cell).

The term “or” may be inclusive or exclusive.

A “genetic modification” refers to any addition, deletion or disruptionto a cell's normal nucleotides. Any method which can achieve the geneticmodification of APCs are within the spirit and scope of this invention.Art recognized methods include viral mediated gene transfer, liposomemediated transfer, transformation, transfection and transduction.

The terms “nucleic acid molecule” or “polynucleotide” will be usedinterchangeably throughout the specification, unless otherwisespecified. As used herein, “nucleic acid molecule” refers to thephosphate ester polymeric form of ribonucleosides (adenosine, guanosine,uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester analogues thereof, such asphosphorothioates and thioesters, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter cilia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of DNA (i.e., the strandhaving a sequence homologous to the mRNA). A “recombinant DNA molecule”is a DNA molecule that has undergone a molecular biologicalmanipulation.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

As used herein, the term “fragment or segment,” as applied to a nucleicacid sequence, gene or polypeptide, will ordinarily be at least about 5contiguous nucleic acid bases (for nucleic acid sequence or gene) oramino acids (for polypeptides), typically at least about 10 contiguousnucleic acid bases or amino acids, more typically at least about 20contiguous nucleic acid bases or amino acids, usually at least about 30contiguous nucleic acid bases or amino acids, preferably at least about40 contiguous nucleic acid bases or amino acids, more preferably atleast about 50 contiguous nucleic acid bases or amino acids, and evenmore preferably at least about 60 to 80 or more contiguous nucleic acidbases or amino acids in length. “Overlapping fragments” as used herein,refer to contiguous nucleic acid or peptide fragments which begin at theamino terminal end of a nucleic acid or protein and end at the carboxyterminal end of the nucleic acid or protein. Each nucleic acid orpeptide fragment has at least about one contiguous nucleic acid or aminoacid position in common with the next nucleic acid or peptide fragment,more preferably at least about three contiguous nucleic acid bases oramino acid positions in common, most preferably at least about tencontiguous nucleic acid bases amino acid positions in common.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least 20nucleotides, more generally at least 23 nucleotides, ordinarily at least26 nucleotides, more ordinarily at least 29 nucleotides, often at least32 nucleotides, more often at least 35 nucleotides, typically at least38 nucleotides, more typically at least 41 nucleotides, usually at least44 nucleotides, more usually at least 47 nucleotides, preferably atleast 50 nucleotides, more preferably at least 53 nucleotides, and inparticularly preferred embodiments will be at least 56 or morenucleotides.

A “vector” is a composition which can transduce, transfect, transform orinfect a cell, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell, or in a manner not nativeto the cell. A cell is “transduced” by a nucleic acid when the nucleicacid is translocated into the cell from the extracellular environment.Any method of transferring a nucleic acid into the cell may be used; theterm, unless otherwise indicated, does not imply any particular methodof delivering a nucleic acid into a cell. A cell is “transformed” by anucleic acid when the nucleic acid is transduced into the cell andstably replicated. A vector includes a nucleic acid (ordinarily RNA orDNA) to be expressed by the cell. A vector optionally includes materialsto aid in achieving entry of the nucleic acid into the cell, such as aviral particle, liposome, protein coating or the like. A “celltransduction vector” is a vector which encodes a nucleic acid capable ofstable replication and expression in a cell once the nucleic acid istransduced into the cell.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, promoters, silencing elements, which induce, inhibit orcontrol transcription of protein coding sequences with which they areoperably linked.

As used herein, the term “downstream” when used in reference to adirection along a nucleotide sequence means in the direction from the 5′to the 3′ end. Similarly, the terms “upstream” means in the directionfrom the 3′ to the 5′ end.

As used herein, the term “gene” means the gene and all currently knownvariants thereof and any further variants which may be elucidated.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype target genes. Variants may result from at least one mutation in thenucleic acid sequence and may result in altered mRNAs or in polypeptideswhose structure or function may or may not be altered. Any given naturalor recombinant gene may have none, one, or many allelic forms. Commonmutational changes that give rise to variants are generally ascribed tonatural deletions, additions, or substitutions of nucleotides. Each ofthese types of changes may occur alone, or in combination with theothers, one or more times in a given sequence.

As used herein, “variant” of polypeptides refers to an amino acidsequence that is altered by one or more amino acid residues. The variantmay have “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties (e.g., replacement of leucinewith isoleucine). More rarely, a variant may have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological activity may be foundusing computer programs well known in the art, for example, LASERGENEsoftware (DNASTAR).

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between subjects of agiven species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base.

The terms, “complementary” or “complements” are used interchangeablythroughout and mean that two sequences are complementary when thesequence of one can bind to the sequence of the other in ananti-parallel sense wherein the 3′-end of each sequence binds to the5′-end of the other sequence and each A, T(U), G, and C of one sequenceis then aligned with a T(U), A, C, and G, respectively, of the othersequence. Normally, the complementary sequence of the oligonucleotidehas at least 80% or 90%, preferably 95%, most preferably 100%,complementarity to a defined sequence. Preferably, alleles or variantsthereof can be identified. A BLAST program also can be employed toassess such sequence identity.

The term “complementary sequence” as it refers to a polynucleotidesequence, relates to the base sequence in another nucleic acid moleculeby the base-pairing rules. More particularly, the term or like termrefers to the hybridization or base pairing between nucleotides ornucleic acids, such as, for instance, between the two strands of adouble stranded DNA molecule or between an oligonucleotide primer and aprimer binding site on a single stranded nucleic acid to be sequenced oramplified. Complementary nucleotides are, generally, A and T (or A andU), or C and G. Two single stranded RNA or DNA molecules are said to besubstantially complementary when the nucleotides of one strand,optimally aligned and compared and with appropriate nucleotideinsertions or deletions, pair with at least about 95% of the nucleotidesof the other strand, usually at least about 98%, and more preferablyfrom about 99% to about 100%. Complementary polynucleotide sequences canbe identified by a variety of approaches including use of well-knowncomputer algorithms and software, for example the BLAST program.

The term “substantial sequence identity”, when used in connection withpeptides/amino acid sequences, refers to peptides/amino acid sequences,which are substantially identical to or similar in sequence, giving riseto a sequence identity in conformation and thus to similar biologicalactivity. The term is not intended to imply a common evolution of thesequences.

Typically, peptides/amino acid sequences having “substantial sequenceidentity” are sequences that are at least 50%, more preferably at least80%, identical in sequence, at least over any regions known to beinvolved in the desired activity. Most preferably, no more than fiveresidues, other than at the termini, are different. Preferably, thedivergence in sequence, at least in the aforementioned regions, is inthe form of “conservative modifications”.

To determine the percent sequence identity of two peptides/amino acidsequences or of two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in one orboth of a first and a second amino acid or nucleic acid sequence foroptimal alignment and non-homologous sequences can be disregarded forcomparison purposes). For example, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence (e.g., when aligning a second sequence to the firstamino acid sequence which has for example 100 amino acid residues, atleast 30, preferably at least 40, more preferably at least 50, even morepreferably at least 60, and even more preferably at least 70, 80 or 90amino acid residues are aligned). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “sequence identity”) The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps, and thelength of each gap, which need to be introduced for optimal alignment ofthe two sequences.

The terms “protein” and “polypeptide” are used interchangeably herein.The term “peptide” is used herein to refer to a chain of two or moreamino acids or amino acid analogs (including non-naturally occurringamino acids), with adjacent amino acids joined by peptide (—NHCO—)bonds. Thus, the peptides of the invention include oligopeptides,polypeptides, proteins, mimetopes and peptidomimetics. Methods forpreparing mimetopes and peptidomimetics are known in the art.

The terms “mimetope” and “peptidomimetic” are used interchangeablyherein. A “mimetope” of a compound X refers to a compound in whichchemical structures of X necessary for functional activity of X havebeen replaced with other chemical structures which mimic theconformation of X. Examples of peptidomimetics include peptidiccompounds in which the peptide backbone is substituted with one or morebenzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science260:1937-1942) and “retro-inverso” peptides (see U.S. Pat. No. 4,522,752to Sisto). The terms “mimetope” and “peptidomimetic” also refer to amoiety, other than a naturally occurring amino acid, thatconformationally and functionally serves as a substitute for aparticular amino acid in a peptide-containing compound without adverselyinterfering to a significant extent with the function of the peptide.Examples of amino acid mimetics include D-amino acids. Peptidessubstituted with one or more D-amino acids may be made using well knownpeptide synthesis procedures. Additional substitutions include aminoacid analogs having variant side chains with functional groups, forexample, b-cyanoalanine, canavanine, djenkolic acid, norleucine,3-phosphoserine, homoserine, dihydroxyphenylalanine,5-hydroxytryptophan, 1-methylhistidine, or 3-methylhistidine

As used herein an “analog” of a compound X refers to a compound whichretains chemical structures of X necessary for functional activity of X,yet which also contains certain chemical structures which differ from X.An example of an analog of a naturally-occurring peptide is a peptidewhich includes one or more non-naturally-occurring amino acids. The term“analog” is also intended to include modified mimetopes and/orpeptidomimetics, modified peptides and polypeptides, and allelicvariants of peptides and polypeptides. Analogs of a peptide willtherefore produce a peptide analog that is substantially homologous or,in other words, has substantial sequence identity to the originalpeptide. The term “amino acid” includes its art recognized meaning.Preferred amino acids include the naturally occurring amino acids, aswell as synthetic derivatives, and amino acids derived from proteins,e.g., proteins such as casein, i.e., casamino acids, or enzymatic orchemical digests of, e.g., yeast, an animal product, e.g., a meatdigest, or a plant product, e.g., soy protein, cottonseed protein, or acorn steep liquor (see, e.g., Traders' Guide to Fermentation Media,Traders Protein, Memphis, Term. (1988), Biotechnology: A Textbook ofIndustrial Microbiology, Sinauer Associates, Sunderland, Mass. (1989),and Product Data Sheet for Corn Steep Liquor, Grain Processing Corp.,IO).

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems which comprise apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

A “heterologous” component refers to a component that is introduced intoor produced within a different entity from that in which it is naturallylocated. For example, a polynucleotide derived from one organism andintroduced by genetic engineering techniques into a different organismis a heterologous polynucleotide that if expressed can encode aheterologous polypeptide. Similarly, a promoter or enhancer that isremoved from its native coding sequence and operably linked to adifferent coding sequence is a heterologous promoter or enhancer.Possible alternative terminology includes “foreign” or “exogenous”. Aheterologous nucleotide sequence may encode a sequence of amino acids,i.e. a peptide or a polypeptide.

A “promoter,” as used herein, refers to a polynucleotide sequence thatcontrols transcription of a gene or coding sequence to which it isoperably linked. A large number of promoters, including constitutive,inducible and repressible promoters, from a variety of differentsources, are well known in the art and are available as or within clonedpolynucleotide sequences (from, e.g., depositories such as the ATCC aswell as other commercial or subject sources).

An “enhancer,” as used herein, refers to a polynucleotide sequence thatenhances transcription of a gene or coding sequence to which it isoperably linked. A large number of enhancers, from a variety ofdifferent sources are well known in the art and available as or withincloned polynucleotide sequences (from, e.g., depositories such as theATCC as well as other commercial or subject sources). A number ofpolynucleotides comprising promoter sequences (such as the commonly-usedCMV promoter) also comprise enhancer sequences.

“Operably linked” refers to a juxtaposition, wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A promoter is operably linked to a coding sequence ifthe promoter controls transcription of the coding sequence. Although anoperably linked promoter is generally located upstream of the codingsequence, it is not necessarily contiguous with it. An enhancer isoperably linked to a coding sequence if the enhancer increasestranscription of the coding sequence. Operably linked enhancers can belocated upstream, within or downstream of coding sequences. Apolyadenylation sequence is operably linked to a coding sequence if itis located at the downstream end of the coding sequence such thattranscription proceeds through the coding sequence into thepolyadenylation sequence.

“Gene delivery,” “gene transfer,” and the like as used herein, are termsreferring to the introduction of an exogenous polynucleotide (sometimesreferred to as a “transgenes”) into a host cell, irrespective of themethod used for the introduction. Such methods include a variety ofwell-known techniques such as vector-mediated gene transfer (by, e.g.,viral infection/transfection, or various other protein-based orlipid-based gene delivery complexes) as well as techniques facilitatingthe delivery of “naked” polynucleotides (such as electroporation, “genegun” delivery and various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

As used herein, a “target cell” or “recipient cell” refers to an subjectcell or cell which is desired to be, or has been, a recipient ofexogenous nucleic acid molecules, polynucleotides and/or proteins. Theterm is also intended to include progeny of a single cell.

“In vivo” gene delivery, gene transfer, gene therapy and the like asused herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

A cell is “transduced” by a nucleic acid when the nucleic acid istranslocated into the cell from the extracellular environment. Anymethod of transferring a nucleic acid into the cell may be used; theterm, unless otherwise indicated, does not imply any particular methodof delivering a nucleic acid into a cell. A cell is “transformed” by anucleic acid when the nucleic acid is transduced into the cell andstably replicated. A vector includes a nucleic acid (ordinarily RNA orDNA) to be expressed by the cell. A vector optionally includes materialsto aid in achieving entry of the nucleic acid into the cell, such as aviral particle, liposome, protein coating or the like. A “celltransduction vector” is a vector which encodes a nucleic acid capable ofstable replication and expression in a cell once the nucleic acid istransduced into the cell.

As used herein, “homologous recombination” means a nucleotide sequenceon one vector is homologous to a nucleotide sequence on another vector.Using restriction enzymes to cut the two sequences and ligating the twosequences results in the two vectors combining. Typically, severalkilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors).

Homologous nucleic acid sequences, when compared, exhibit significantsequence identity or similarity. The standards for sequence identity innucleic acids are either measures for sequence identity generally usedin the art by sequence comparison or based upon hybridizationconditions. The hybridization conditions are described in greater detailbelow.

Sequence homology and sequence identity are used interchangeably herein.

“Stringency” is meant the combination of conditions to which nucleicacids are subject that cause the duplex to dissociate, such astemperature, ionic strength, and concentration of additives such asformamide. Conditions that are more likely to cause the duplex todissociate are called “higher stringency”, e.g. higher temperature,lower ionic strength and higher concentration of formamide.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.10 M NaCl at temperatures ofabout 50° C. to about 70° C.

For certain applications, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated depending on the desired results.

The phrase “hybridizing conditions” and its grammatical equivalents,when used with a maintenance time period, indicates subjecting thehybridization reaction admixture, in context of the concentration of thereactants and accompanying reagents in the admixture, to time,temperature, pH conditions sufficient to allow the polynucleotide probeto anneal with the target sequence, typically to form the nucleic acidduplex. Such time, temperature and pH conditions required to accomplishthe hybridization depend, as is well known in the art on the length ofthe polynucleotide probe to be hybridized, the degree of complementaritybetween the polynucleotide probe and the target, the guanidine andcytosine content of the polynucleotide, the stringency of thehybridization desired, and the presence of salts or additional reagentsin the hybridization reaction admixture as may affect the kinetics ofhybridization. Methods for optimizing hybridization conditions for agiven hybridization reaction admixture are well known in the art.

As used herein, “substantial sequence identity” in the nucleic acidsequence comparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least 56%, moregenerally at least 59%, ordinarily at least 62%, more ordinarily atleast 65%, often at least 68%, more often at least 71%, typically atleast 74%, more typically at least 77%, usually at least 80%, moreusually at least about 85%, preferably at least about 90%, morepreferably at least about 95 to 98% or more, and in particularembodiments, as high at about 99% or more of the nucleotides.Alternatively, substantial sequence identity exists when the segmentswill hybridize under selective hybridization conditions, to a strand, orits complement, typically using a fragment derived from SEQ ID NO: 1.Typically, selective hybridization will occur when there is at leastabout 55% sequence identity over a stretch of at least about 14nucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. See Kanehisa (1984)Nuc. Acids Res. 12:203-213. The length of sequence identity comparison,as described, may be over longer stretches, and in certain embodimentswill be over a stretch of at least about 17 nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides. The endpointsof the segments may be at many different pair combinations. Indetermining sequence identity or percent homology the below discussedprotocols and programs for sequence similarity are suitably employedincluding the BLAST algorithm.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion (e.g., allelic variant) thereof. A portion of agene of which there are at least two different forms, i.e., twodifferent nucleotide sequences, is referred to as a “polymorphic regionof a gene”. A specific genetic sequence at a polymorphic region of agene is an allele. A polymorphic region can be a single nucleotide, theidentity of which differs in different alleles. A polymorphic region canalso be several nucleotides long.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to identify, for example, other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to NIP2b, NIP2cL, and NIP2cS nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to NIP2b, NIP2cL, and NIP2cS protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

Sequence similarity searches can be also performed manually or by usingseveral available computer programs known to those skilled in the art.Preferably, Blast and Smith-Waterman algorithms, which are available andknown to those skilled in the art, and the like can be used. Blast isNCBI's sequence similarity search tool designed to support analysis ofnucleotide and protein sequence databases. The GCG Package provides alocal version of Blast that can be used either with public domaindatabases or with any locally available searchable database. GCG Packagev9.0 is a commercially available software package that contains over 100interrelated software programs that enables analysis of sequences byediting, mapping, comparing and aligning them. Other programs includedin the GCG Package include, for example, programs which facilitate RNAsecondary structure predictions, nucleic acid fragment assembly, andevolutionary analysis. In addition, the most prominent genetic databases(GenBank, EMBL, PIR, and SWISS-PROT) are distributed along with the GCGPackage and are fully accessible with the database searching andmanipulation programs. GCG can be accessed through the Internet at, forexample, www.gcg.com/. Fetch is a tool available in GCG that can getannotated GenBank records based on accession numbers and is similar toEntrez. Another sequence similarity search can be performed withGeneWorld and GeneThesaurus from Pangea. GeneWorld 2.5 is an automated,flexible, high-throughput application for analysis of polynucleotide andprotein sequences. GeneWorld allows for automatic analysis andannotations of sequences. Like GCG, GeneWorld incorporates several toolsfor sequence identity searching, gene finding, multiple sequencealignment, secondary structure prediction, and motif identification.GeneThesaurus 1.0™ is a sequence and annotation data subscriptionservice providing information from multiple sources, providing arelational data model for public and local data.

Another alternative sequence similarity search can be performed, forexample, by BlastParse. BlastParse is a PERL script running on a UNIXplatform that automates the strategy described above. BlastParse takes alist of target accession numbers of interest and parses all the GenBankfields into “tab-delimited” text that can then be saved in a “relationaldatabase” format for easier search and analysis, which providesflexibility. The end result is a series of completely parsed GenBankrecords that can be easily sorted, filtered, and queried against, aswell as an annotations-relational database.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule to hybridizeto at least approximately 6 consecutive nucleotides of a sample nucleicacid.

“Substantially purified” refers to nucleic acid molecules or proteinsthat are removed from their natural environment and are isolated orseparated, and are at least about 60% free, preferably about 75% free,and most preferably about 90% free, from other components with whichthey are naturally associated.

An “antigen” is any substance that reacts specifically with antibodiesor T lymphocytes (T cells). An “antigen-binding site” is the part of animmunoglobulin molecule that specifically binds an antigen.Additionally, an antigen-binding site includes any such site on anyantigen-binding molecule, including, but not limited to, an MHC moleculeor T cell receptor. “Antigen processing” refers to the degradation of anantigen into fragments (e.g., the degradation of a protein intopeptides) and the association of one or more of these fragments (e.g.,via binding) with MHC molecules for presentation by “antigen-presentingcells” to specific T cells.

“Dendritic cells” (DC) are potent antigen-presenting cells, capable oftriggering a robust adaptive immune response in vivo. It has been shownthat activated, mature DC provide the signals required for T cellactivation and proliferation. These signals can be categorized into twotypes. The first type, which gives specificity to the immune response,is mediated through interaction between the T-cell receptor/CD3(“TCR/CD3”) complex and an antigenic peptide presented by a majorhistocompatibility complex (“MHC” defined above) class I or II proteinon the surface of APCs. The second type of signal, called aco-stimulatory signal, is neither antigen-specific nor MHC-restricted,and can lead to a full proliferation response of T cells and inductionof T cell effector functions in the presence of the first type ofsignals. This two-fold signaling can, therefore, result in a vigorousimmune response. As noted supra, in most non-avian vertebrates, DC arisefrom bone marrow-derived precursors. Immature DC are found in theperipheral blood and cord blood and in the thymus. Additional immaturepopulations may be present elsewhere. DC of various stages of maturityare also found in the spleen, lymph nodes, tonsils, and human intestine.Avian DC may also be found in the bursa of Fabricius, a primary immuneorgan unique to avians. In a preferred embodiment, the dendritic cellsof the present invention are mammalian, preferably human, mouse, or rat.

A “co-stimulatory molecule” encompasses any single molecule orcombination of molecules which, when acting together with a peptide/MHCcomplex bound by a T cell receptor on the surface of a T cell, providesa co-stimulatory effect which achieves activation of the T cell thatbinds the peptide.

As used herein, “immunoreceptors” will refer to class I MHC(HLA-A, -B,-C, -G) and the like) and other immune related receptors, such as forexample Gp49, PIR, PIRA, PIRB, LIR, NKR-P1, NKp46, Digr1, ILT, MIR, KIRand the like. MHC may also include other classes such as MHC class IIand MHC class III, derivatives and mutants thereof. The human MHCcomplex is also called the human leukocyte antigen (HLA) complex. MHCantigens are divided into MHC class I antigens (in humans, this classincludes HLA-A, -B, and -C antigens) and MHC class II antigens (inhumans, this class includes HLA-DP, -DQ, and -DR antigens). Thus, theterms “MHC-II antigens”, “MHC class II antigens”, and “MHC class IItransplantation antigens” are used interchangeably herein to refer tothe class of proteins, which in humans, includes HLA-DP, -DQ and -DRantigens. While the terms “MHC class II genes” and “MHC-II genes” areused interchangeably herein to refer to the genes which encode the MHCclass II transplantation antigens. The term “MHC-II” is used herein torefer to the gene locus which encodes the MHC class II transplantationantigens, as well as the group of proteins encoded by that locus.Transplantation antigens also include cell surface molecules other thanMHC class I and II antigens. These antigens include the following: (1)the ABO antigens involved in blood cell recognition; (2) cell adhesionmolecules such as ICAM, which is involved in leukocyte cell-cellrecognition; and (3) β2-microglobulin, a polypeptide associated with the44 kd heavy chain polypeptide that comprises the HLA-I antigens but isnot encoded by the MHC complex. HLA haplotypes/allotypes vary fromsubject to subject and it is often helpful to determine the subject'sHLA type. The HLA type may be determined via standard typing proceduresand the peripheral blood lymphocytes (PBLs) purified by Ficollgradients.

“Diagnostic” or “diagnosed” means identifying the presence or nature ofa pathologic condition. Diagnostic methods differ in their sensitivityand specificity. The “sensitivity” of a diagnostic assay is thepercentage of diseased subjects who test positive (percent of “truepositives”). Diseased subjects not detected by the assay are “falsenegatives.” Subjects who are not diseased and who test negative in theassay, are termed “true negatives.” The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the “false positive”rate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

The terms “subject” or “subject” are used interchangeably herein, and ismeant a mammalian subject to be treated, with human subjects beingpreferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary application, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters; and primates.

“Label molecules” are chemical or biochemical moieties used for labelinga polynucleotide, a polypeptide, or an antibody. They include, but arenot limited to, radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chromogenic agents, chemiluminescentagents, magnetic particles, and the like. Reporter moleculesspecifically bind, establish the presence of, and allow quantificationof a particular polynucleotide, polypeptide, or antibody.

“Sample” is used herein in its broadest sense. A sample comprisingpolynucleotides, polypeptides, peptides, antibodies and the like maycomprise a bodily fluid; a soluble fraction of a cell preparation, ormedia in which cells were grown; a chromosome, an organelle, or membraneisolated or extracted from a cell; genomic DNA, RNA, or cDNA,polypeptides, or peptides in solution or bound to a substrate; a cell; atissue; a tissue print; a fingerprint, skin or hair; and the like.

As used herein, “fresh tumors” refer to tumors removed from a host bysurgical or other means.

As used herein, “proliferative growth disorder, “neoplastic disease,”“tumor”, “cancer” are used interchangeably as used herein refers to acondition characterized by uncontrolled, abnormal growth of cells.Preferably the cancer to be treated is MUC-1 positive cancer and theabnormal proliferation of cells can be any cell in the organ. Examplesof cancer include but are not limited to, carcinoma, blastoma, andsarcoma. As used herein, the term “carcinoma” refers to a new growththat arises from epithelium, found in skin or, more commonly, the liningof body organs.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. “Treatment” may also bespecified as palliative care. Those in need of treatment include thosealready with the disorder as well as those in which the disorder is tobe prevented. In tumor (e.g., cancer) treatment, a therapeutic agent maydirectly decrease the pathology of tumor cells, or render the tumorcells more susceptible to treatment by other therapeutic agents, e.g.,radiation and/or chemotherapy.

The term “in need of such treatment” as used herein refers to a judgmentmade by a care giver such as a physician, nurse, or nurse practitionerin the case of humans that a subject requires or would benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a care giver's expertise, but that include the knowledgethat the subject is ill, or will be ill, as the result of a conditionthat is treatable by the compositions of the invention.

“Cells of the immune system” or “immune cells” as used herein, is meantto include any cells of the immune system that may be assayed,including, but not limited to, B lymphocytes, also called B cells, Tlymphocytes, also called T cells, natural killer (NK) cells,lymphokine-activated killer (LAK) cells, monocytes, macrophages,neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stemcells, dendritic cells, peripheral blood mononuclear cells,tumor-infiltrating (TIL) cells, gene modified immune cells includinghybridomas, drug modified immune cells, and derivatives, precursors orprogenitors of the above cell types.

“Immune effector cells” refers to cells capable of binding an antigenand which mediate an immune response. These cells include, but are notlimited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes,macrophages, natural killer (NK) cells and cytotoxic T lymphocytes(CTLs), for example CTL lines, CTL clones, and CTLs from tumor,inflammatory, or other infiltrates.

“T cells” or “T lymphocytes” are a subset of lymphocytes originating inthe thymus and having heterodimeric receptors associated with proteinsof the CD3 complex (e.g., a rearranged T cell receptor, theheterodimeric protein on the T cell surfaces responsible for antigen/MHCspecificity of the cells). T cell responses may be detected by assaysfor their effects on other cells (e.g., target cell killing, macrophage,activation, B-cell activation) or for the cytokines they produce.

The term “activated T cell,” as used herein, refers to a T cell thatexpresses antigens indicative of T-cell activation (that is, T cellactivation markers). Examples of T cell activation markers include, butare not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS,OX-40 and 4-1BB. The expression of activation markers can be measured bytechniques known to those of skill in the art, including, for example,western blot analysis, northern blot analysis, RT-PCR,immunofluorescence assays, and fluorescence activated cell sorter (FACS)analysis.

The term “resting T cell,” as used herein, refers to a T cell that doesnot express T-cell activation markers. Resting T cells include, but arenot limited to, T cells which are CD25⁻, CD69⁻, ICOS⁻, SLAM⁻, and4-1BB⁻. The expression of these markers can be measured by techniquesknown to those of skill in the art, including, for example, western blotanalysis, northern blot analysis, RT-PCR, immunofluorescence assays, andfluorescence activated cell sorter (FACS) analysis.

“CD4” is a cell surface protein important for recognition by the T cellreceptor of antigenic peptides bound to MHC class II molecules on thesurface of an APC. Upon activation, naïve CD4 T cells differentiate intoone of at least two cell types, Th1 cells and Th2 cells, each type beingcharacterized by the cytokines it produces. “Th1 cells” are primarilyinvolved in activating macrophages with respect to cellular immunity andthe inflammatory response, whereas “Th2 cells” or “helper T cells” areprimarily involved in stimulating B cells to produce antibodies (humoralimmunity). CD4 is the receptor for the human immunodeficiency virus(HIV). Effector molecules for Th1 cells include, but are not limited to,IFN-γ, GM-CSF, TNF-α, CD40 ligand, Fas ligand, IL-3, TNF-β, and IL-2.Effector molecules for Th2 cells include, but are not limited to, IL-4,IL-5, CD40 ligand, IL-3, GS-CSF, IL-10, TGF-β, and eotaxin. Activationof the Th1 type cytokine response can suppress the Th2 type cytokineresponse.

“CD8” is a cell surface protein important for recognition by the T cellreceptor of antigenic peptides bound to MHC class 1 molecules. CD8 Tcells usually become “cytotoxic T cells” or “killer T cells” andactivate macrophages. Effector molecules include, but are not limitedto, perforin, granzymes, Fas ligand, IFN-γ, TNF-α, and TNF-β.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope “A”, the presence of a proteincomprising epitope A (or free, unlabeled A) in a reaction comprisinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody. “Specific binding” in general, refers to any immunerelated molecule binding to its ligand, such as for example the bindingof a T cell receptor expressed by a T lymphocyte, to an MHC molecule andpeptide on an antigen presenting cell.

“Activity,” “activation,” or “augmentation” is the ability of immunecells to respond and exhibit, on a measurable level, an immune function.Measuring the degree of activation refers to a quantitative assessmentof the capacity of immune cells to express enhanced activity whenfurther stimulated as a result of prior activation. The enhancedcapacity may result from biochemical changes occurring during theactivation process that allow the immune cells to be stimulated toactivity in response to low doses of stimulants.

Immune cell activity that may be measured include, but is not limitedto, (1) cell proliferation by measuring the cell or DNA replication; (2)enhanced cytokine production, including specific measurements forcytokines, such as IFN-γ, GM-CSF, or TNF-α; (3) cell mediated targetkilling or lysis; (4) cell differentiation; (5) immunoglobulinproduction; (6) phenotypic changes; (7) production of chemotacticfactors or chemotaxis, meaning the ability to respond to a chemotactinwith chemotaxis; (8) immunosuppression, by inhibition of the activity ofsome other immune cell type; and, (9) apoptosis, which refers tofragmentation of activated immune cells under certain circumstances, asan indication of abnormal activation.

An “adjuvant” is any substance capable of enhancing the immune responseto an antigen with which it is mixed. Depending on the host species,various adjuvants may be used to increase immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, and dinitrophenol, as well as BCG (bacilli Calmette-Guerin) andCorynabacterium parvum, which are often used in humans, and ligands ofCCR6 and other chemokine receptors.

A “chemokine” is a small cytokine involved in the migration andactivation of cells, including phagocytes and lymphocytes, and plays arole in inflammatory responses. Three classes of chemokines have beendefined by the arrangement of the conserved cysteine (C) residues of themature proteins: the CXC or a chemokines that have one amino acidresidue separating the first two conserved cysteine residues; the CC orβ chemokines in which the first two conserved cysteine residues areadjacent; the C or γ chemokines which lack two (the first and third) ofthe four conserved cysteine residues. Within the CXC subfamily, thechemokines can be further divided into two groups. One group of the CXCchemokines have the characteristic three amino acid sequence ELR(glutamic acid-leucine-arginine) motif immediately preceding the firstcysteine residue near the amino terminus. A second group of CXCchemokines lack such an ELR domain. The CXC chemokines with the ELRdomain (including IL-8, GROα/β/γ, mouse KC, mouse MIP-2, ENA-78, GCP-2,PBP/CTAPIII/β-TG/NAP-2) act primarily on neutrophils as chemoattractantsand activators, inducing neutrophil degranulation with release ofmyeloperoxidase and other enzymes. The CXC chemokines without the ELRdomain (e.g., IP-10/mouse CRG, Mig, PBSF/SDF-1, PF4), the CC chemokines(e.g., MIP-1α, MIP-1 β, RANTES, MCP-1/2/3/4/mouse JE/mouse MARC,eotaxin, I-309/TCA3, HCC-1, C10), and the C chemokines (e.g.,lymphotactin), chemoattract and activate monocytes, dendritic cells,T-lymphocytes, natural killer cells, B-lymphocytes, basophils, andeosinophils.

A “cytokine” is a protein made by a cell that affect the behavior ofother cells through a “cytokine receptor” on the surface of the cellsthe cytokine effects. Cytokines manufactured by lymphocytes aresometimes termed “lymphokines.” Examples of cytokines includeinterleukins, interferons and the like.

By “immunologically effective” is meant an amount of the peptide orfragment thereof which is effective to activate an immune response toprevent or treat proliferative cell growth disorders, such as cancer.Obviously, such amounts will vary between species and subjects dependingon many factors. For example, higher doses will generally be requiredfor an effective immune response in a human compared with a mouse.

As used herein, the term “polypeptide” encompasses amino acid chains ofany length, including full length proteins containing the sequencesrecited herein. A polypeptide comprising an epitope of a proteincontaining a sequence as described herein may consist entirely of theepitope, or may contain additional sequences. The additional sequencesmay be derived from the native protein or may be heterologous, and suchsequences may (but need not) possess immunogenic or antigenicproperties.

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, wherean antibody binding domain is formed from the folding of variabledomains of an antibody molecule to form three-dimensional binding spaceswith an internal surface shape and charge distribution complementary tothe features of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. Antibodies include recombinantproteins comprising the binding domains, as wells as fragments,including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments. The teen “antibody,”as used herein, also includes antibody fragments either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies. It also includes polyclonal antibodies,monoclonal antibodies, chimeric antibodies, humanized antibodies, orsingle chain antibodies. “Fc” portion of an antibody refers to thatportion of an immunoglobulin heavy chain that comprises one or moreheavy chain constant region domains, CH_(I), CH₂ and CH₃, but does notinclude the heavy chain variable region.

An “epitope”, as used herein, is a portion of a polypeptide that isrecognized (i.e., specifically bound) by a B-cell and/or T-cell surfaceantigen receptor. Epitopes may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides derived from the nativepolypeptide for the ability to react with antigen-specific antiseraand/or T-cell lines or clones. An epitope of a polypeptide is a portionthat reacts with such antisera and/or T-cells at a level that is similarto the reactivity of the full length polypeptide (e.g., in an FT ISAand/or T-cell reactivity assay). Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may alsobe predicted via computer analysis. Polypeptides comprising an epitopeof a polypeptide that is preferentially expressed in a tumor tissue(with or without additional amino acid sequence) are within the scope ofthe present invention.

As used herein, the term “agonist polypeptide” refers to epitopes in thepolypeptide which activate a stronger immune response than a nativepolypeptide. Examples of differences in properties between an agonistpolypeptide versus a native polypetide include, but not limited to a)binding HLA molecules at lower peptide concentrations, (b) demonstrate ahigher avidity for HLA molecules in dissociation assays, (c) when usedwith antigen-presenting cells induce the production of more IFN-γ by Tcells derived with the use of the native peptide. Increased or augmentedimmune response are measured as described above.

As used herein, “native polypeptide” refers to a polypeptide as found inits natural environment. For example, a native MUC-1 tumor antigen isexpressed by a tumor cell in a subject.

In a preferred embodiment, agonist polypeptides generate stronger immuneresponses, as compared to the native polypeptide. For example, comparedwith the native P-92 peptide, agonist polypeptides (a) bind HLA-A2 atlower peptide concentrations, (b) demonstrate a higher avidity forHLA-A2 in dissociation assays, (c) when used with antigen-presentingcells induce the production of more IFN-γ by T cells derived with theuse of the native peptide, and (d) were capable of more efficientlygenerating MUC-1-specific human T-cell lines from normal volunteers andpancreatic cancer subjects. Most importantly, the T-cell lines generatedusing the agonist epitope were more efficient than those generated withthe native epitope, in the lysis of targets pulsed with the nativeepitope and in the lysis of HLA-A2 human tumor cells expressing MUC-1.

In another preferred embodiment, subjects suffering from or susceptibleto tumors, infectious diseases and the like are treated with autologousantigen presenting cells, such as for example dendritic cells (DCs),that have been transduced with a viral vector encoding anyone of thepolypeptides as identified by SEQ ID NO: 1 through 19, fragments orvariants thereof, optionally expressing co-stimulatory molecules. Forexample, autologous DCs infected with rF-MUC-1/TRICOM were used as APC.rF-MUC-1/TRICOM is a replication-defective avipox vector containing thetransgenes for MUC-1 and for a triad of human costimulatory molecules(B7-1, ICAM-1 and LFA-3, designated TRICOM). rF-MUC-1/TRICOM was shownto efficiently infect human DCs and hyperexpress each of thecostimulatory molecules, as well as MUC-1, on the DC surface (Table 2).

In another preferred embodiment, the invention provides a method forgenerating an immune response to a weakly immunogenic antigen comprisingadministering to an subject an agonist polypeptide, as identified by anyone of SEQ ID NO: 1 through 19, variants or fragments thereof, with ahigh avidity for HLA fused to the weak immunogen.

In a preferred embodiment, the invention provides an isolated nucleicacid molecule which encodes an agonist polypeptide antigen derived froma tumor antigen, such as for example, MUC-1, wherein the agonistpolypeptide stimulates a stronger immune response as compared to anative polypeptide. Other examples of tumor antigens, include, but arenot limited to HER2/neu, carcinoembryonic antigen (CEA), p53.

In another preferred embodiment, the invention provides a nucleic acidmolecule comprising a nucleic acid sequence corresponding to any one ofthe amino acid sequences as identified by SEQ ID NO: 1 through 19,fragments or variants thereof. SEQ ID NO: 1 through 19 are identified by

SEQ ID SEQ ID NO Peptide NO (peptide) sequence Nucleotide sequence(n.t.) 1 ATWGQDVTSV GCC/ACC/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 20 2ALWGQDVTSV GCC/CTG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 21 3 ALLVLVCVLVGCC/CTG/CTG/GTC/CTG/GTC/TGC/GTC/CTG/GTC 22 4 TISDVSVSDVACC/ATC/TCG/GAT/GTC/TCG/GTC/TCG/GAT/GTC 23 5 ALAIVYLIALGCC/CTG/GCC/ATC/GTC/TAC/CTG/ATC/GCC/CTG 24 6 VLVALAIVYLGTC/CTG/GTC/GCC/CTG/GCC/ATC/GTC/TAC/CTG 25 7 YLIALAVCQCTAC/CTG/ATC/GCC/CTG/GCC/GTC/TGC/CAA/TGC 26 8 WGQDVTSVPVTGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC/CCA/GTC 27 9 REGTINVHDVAGA/GAA/GGT/ACC/ATC/AAC/GTC/CAC/GAT/GTC 28 10 GTQSPFFLLLGGC/ACC/CAG/TCT/CCT/TTC/TTC/CTG/CTG/CTG 29 11 LAFREGTINVCTG/GCC/TTC/AGA/GAA/GGT/ACC/ATC/AAC/GTC 30 12 TLASHSTKTDACT/CTG/GCC/TCG/CAC/TCG/ACC/AAG/ACC/GAT 31 13 LQRDISEMFLCTG/CAA/AGA/GAT/ATC/TCG/GAA/ATG/TTC/CTG 32 14 AIWGQDVTSVGCC/ACT/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 33 15 ALWGQDVTSLGCC/CTG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/CTG 34 16 AMWGQDVTSVGCC/ATG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/GTC 35 17 AMWGQDVTSLGCC/ATG/TGG/GGA/CAG/GAT/GTC/ACC/TCG/CTG 36 18 AIWGQDVTSLGCC/ACT/TGG/GGAJCAG/GAT/GTC/ACC/TCG/CTG 37 19 ALWGQDVTSV

In another preferred embodiment, the invention provides for a vectorcomprising an isolated nucleic acid molecule expressing any one of aminoacids identified by SEQ ID NO: 1 through 19, fragments or variants,thereof. The vector preferably encodes encoding immune cellco-stimulatory molecules, such as for example, B7-1, ICAM-1 and LFA-3.

In yet another preferred embodiment, the invention provides for thetransduction of dendritic cells with a vector comprising any one of themolecules as identified by SEQ ID NO: 1 through 19, fragments orvariants thereof, and optionally, immune cell co-stimulatory molecules,such as for example, B7-1, ICAM-1 and LFA-3. These recombinant vectorsprovide specific anti-tumor effect for subjects who have been diagnosedwith MUC-1⁺ tumors. However, this antigen is merely an illustrativeexample and is not meant to be construed as limiting in any way.Examples of other antigens that are useful for treating different typesof cancers, include, but not limited to overexpressed or mutated formsof antigens. For example, Her2/neu⁺ tumors such as breast, renal,prostate, and other HER2 tumors, carcinogenic embryonic antigen (CEA)for gastro-intestinal cancers; K-ras for lung, gastrointestinal andbladder cancers; p53 which affects a wide variety of neoplastic growth;SARDT3 in neck and head cancers.

In another preferred embodiment, dendritic cells of an subject,suffering from or susceptible to, cancer, are transduced in vivo withrecombinant vectors expressing agonist polypeptide epitopes. Dendriticcells can be isolated from a subject, cultured ex vivo with a vector,and then re-infusing the cultured dendritic cells into the subject.Culturing of dendritic cells is described in detail in the exampleswhich follow. Alternatively, the vector may be administered to a subjectin need of such treatment.

In a preferred embodiment, transduced dendritic cells present antigen,for example, agonist peptide fragments of the MUC-1 antigen on theirsurface. Lymphocytes, specific for the presented antigens, areactivated, proliferate and recognize tumor cells expressing the MUC-1antigen. Lymphocytes include, B cells, T helper cells and cytotoxic Tcells. Recognition, of any cell expressing antigenic epitopes by theimmune cells, results in the destruction of a tumor cell.

In another preferred embodiment, the invention provides a nucleic acidvector comprising one or more nucleic acid sequences encodingpolypeptides as identified by any one of SEQ ID NO: 1 through 19,fragments or variants thereof, operably linked to an inducible promoter.

In another preferred embodiment the nucleic acid vector is a viralvector, plasmid and the like. Preferably the nucleic acid vectorcomprises an inducible promoter which is tissue specific, andoptionally, immune cell co-stimulatory molecules.

In another preferred embodiment, the vector comprising a nucleic acidsequence encoding any one of the polypeptides identified by SEQ ID NO: 1through 19.

In another preferred embodiment, the vector codes for any one of thepolypeptides identified by any one of SEQ ID NO: 1 through 19 having asequence identity to anyone one of SEQ ID NO: 1 through 19 of at leastabout 10%, more preferably, 25%, even more preferably about 40%, 50%,60%, 70%, 80%, 90%, or 99.9%.

In another preferred embodiment, the invention provides a host cellexpressing the polypeptide products of the vector as identified by anyone of SEQ ID NO: 1 through 19 having a sequence identity to anyone oneof SEQ ID NO: 1 through 19 of at least about 10%, more preferably, 25%,even more preferably about 40%, 50%, 60%, 70%, 80%, 90%, or 99.9%.Preferably the host cell is an antigen presenting cell, such as forexample, a monocyte/macrophage, dendritic cell or the like.

In accordance with the invention, the transduced dendritic cells presentantigen to cells of the immune system and activate the immune system torecognize tumor antigen epitopes, such as for example a tumor cellexpressing the MUC-1 antigen.

In a preferred embodiment, the vector is a avipox vector comprisingnucleic acid molecules encoding agonist polypeptides and co-stimulatorymolecules, as described in detail in the examples which follow. Othervectors may also be used. Preferred vectors include viral vectors,fusion proteins and chemical conjugates. Retroviral vectors includemoloney murine leukemia viruses. DNA viral vectors are preferred. Viralvectors can be chosen to introduce the genes to cells of choice. Suchvectors include pox vectors such as orthopox or avipox vectors,herpesvirus vectors such as herpes simplex I virus (HSV) vector (Gelleret al., 1995, J. Neurochem., 64:487; Lim et al., 1995, in DNA Cloning:Mammalian Systems, D. Glover, ed., Oxford Univ. Press, Oxford, England;Geller et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 1149), otheradenovirus vectors (LeGal LaSalle et al., 1993, Science 259: 988;Davidson et al., 1993, Nat. Genet. 3: 219; Yang et al., 1995, J. Virol.69: 2004) and adeno-associated virus vectors (Kaplitt et al., 1994, Nat.Genet. 8: 148; Kotin, et al. WO 98/11244 (Mar. 19, 1998) and Chiorini,et al WO 99/61601 (Dec. 2, 1999)).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only short term expression of the nucleic acid.Adenovirus vectors, adeno-associated virus vectors and herpes simplexvirus vectors are preferred for introducing the nucleic acid into neuralcells. The adenovirus vector results in a shorter term expression (about2 months) than adeno-associated virus (about 4 months), which in turn isshorter than HSV vectors. The vectors can be introduced by standardtechniques, e.g. infection, transfection, transduction ortransformation. Examples of modes of gene transfer include for example,naked DNA calcium phosphate precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection andviral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Other methods thatcan be used include catheters, intravenous, parenteral, intraperitoneal,and subcutaneous injection, and oral or other known routes ofadministration.

Another preferred method is DNA immunization. DNA immunization employsthe subcutaneous injection of a plasmid DNA (pDNA) vector encoding atumor marker. The pDNA sequence is taken up by antigen presenting cells(APC), preferably by dendritic cells. Once inside the cell, the DNAencoding protein is transcribed and translated and presented tolymphocytes.

Genetic constructs comprise a nucleotide sequence that encodes thenucleic acid sequence of choice and preferably includes an intracellulartrafficking sequence operably linked to regulatory elements needed forgene expression.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material in the form of a plasmid orplasmids. Alternatively, linear DNA which can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents which promote DNA integration into chromosomes may beadded. DNA sequences which are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal.

Genetic constructs include regulatory elements necessary for geneexpression of a nucleic acid molecule. The elements include: a promoter,an initiation codon, a stop codon, and a polyadenylation signal. Inaddition, enhancers may be required for gene expression of the sequenceof choice, for example, the agonist polypeptides identified by SEQ IDNO: 1 through 19, variants or fragments thereof. It is necessary thatthese elements be operably linked to the sequence that encodes thedesired proteins and that the regulatory elements are operable in thesubject to whom they are administered.

Initiation codons and stop codons are generally considered to be part ofa nucleotide sequence that encodes the immunogenic target protein.However, it is necessary that these elements are functional in thesubject to whom the gene construct is administered. The initiation andtermination codons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the subject.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metallothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. For example, plasmidspCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the EpsteinBarr virus origin of replication and nuclear antigen EBNA-1 codingregion which produces high copy episomal replication withoutintegration.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs which are functional in the cells.

The method of the present invention comprises the steps of administeringnucleic acid molecules to tissue of the subject. In some preferredembodiments, the nucleic acid molecules are administeredintramuscularly, intranasally, intraperitoneally, subcutaneously,intradermally, or topically or by lavage to mucosal tissue selected fromthe group consisting of vaginal, rectal, urethral, buccal andsublingual.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a facilitating agent. Facilitatingagents are also referred to as polynucleotide function enhancers orgenetic vaccine facilitator agents. Facilitating agents are described ine.g. International Application No. PCT/US94/00899 filed Jan. 26, 1994and International Application No. PCT/US95/04071 filed Mar. 30, 1995,both incorporated herein by reference. Facilitating agents which areadministered in conjunction with nucleic acid molecules may beadministered as a mixture with the nucleic acid molecule or administeredseparately simultaneously, before or after administration of nucleicacid molecules.

In some preferred embodiments, the genetic constructs of the inventionare formulated with or administered in conjunction with a facilitatorselected from the group consisting of, for example, benzoic acid esters,anilides, amidines, urethans and the hydrochloride salts thereof such asthose of the family of local anesthetics. The facilitating agent isadministered prior to, simultaneously with or subsequent to the geneticconstruct. The facilitating agent and the genetic construct may beformulated in the same composition.

In some embodiments of the invention, the subject is first subject toinjection of the facilitator prior to administration of the geneticconstruct. That is, for example, up to about a week to ten days prior toadministration of the genetic construct, the subject is first injectedwith the facilitator. In some embodiments, the subject is injected withthe facilitator about 1 to 5 days; in some embodiments 24 hours, beforeor after administration of the genetic construct. Alternatively, if usedat all, the facilitator is administered simultaneously, minutes beforeor after administration of the genetic construct. Accordingly, thefacilitator and the genetic construct may be combined to form a singlepharmaceutical composition.

In some embodiments, the genetic constructs are administered free offacilitating agents, that is in formulations free from facilitatingagents using administration protocols in which the genetic constructionsare not administered in conjunction with the administration offacilitating agents.

Nucleic acid molecules which are delivered to cells according to theinvention may serve as genetic templates for proteins that function asprophylactic and/or therapeutic immunizing agents. In preferredembodiments, the nucleic acid molecules comprise the necessaryregulatory sequences for transcription and translation of the codingregion in the cells of the animal.

In further embodiments of the present invention, the agonistpolypeptides described herein may be used for the immunotherapy of MUC-1positive tumors. In these embodiments, the compounds (which may bepolypeptides, antibodies or nucleic acid molecules) are preferablyincorporated into pharmaceutical compositions or vaccines.Pharmaceutical compositions comprise one or more such compounds and aphysiologically acceptable carrier. Vaccines may comprise one or morepolypeptides and an immune response enhancer, such as an adjuvant or aliposome (into which the compound is incorporated). Pharmaceuticalcompositions and vaccines may additionally contain a delivery system,such as biodegradable microspheres which are disclosed, for example, inU.S. Pat. Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions andvaccines within the scope of the present invention may also containother compounds, including one or more separate polypeptides.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g.,polylactate polyglycolate) may also be employed as carriers for thepharmaceutical compositions of this invention.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to nonspecifically enhance the immune response. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.), alum, biodegradablemicrospheres, monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

In another preferred embodiment, the invention provides a method fortreating a subject suffering from or susceptible to a MUC-1 tumorcomprising administering to a subject any one of the peptides identifiedby SEQ ID NO: 1 through 19, fragments or variants thereof.

In accordance with the invention, an immune response to a MUC-1 tumorantigen, is generated, by administering an agonist polypeptides in atherapeutically effective dose sufficient to generate a cellular immuneresponse, wherein the agonist polypeptides are any one of polypeptidesidentified by SEQ ID NO: 1 through 19, fragments or variants thereof,and optionally immune cell co-stimulatory molecules. Preferably, thepolypeptides as identified by any one of SEQ ID NO: 1 through 19 havinga sequence identity to anyone one of SEQ ID NO: 1 through 19 of at leastabout 10%, more preferably, 25%, even more preferably about 40%, 50%,60%, 70%, 80%, 90%, or 99.9%.

The peptides are administered to an subject suffering from orsusceptible to cancers. Definite clinical diagnosis of a particularcancer warrants the administration of the peptides, including the earlystages of the disease. Prophylactic applications are warranted in caseswhere subjects with familial history of disease and predicted to be atrisk by reliable prognostic indicators could be treated prophylacticallyto interdict cancer prior to onset, such as MUC-1 positive cancer; orcan be administered post operatively.

Peptide vaccines can be administered in many possible formulations, inpharmacologically acceptable mediums. In the case of a short peptide,the peptide can be conjugated to a carrier, such as KLH, in order toincrease its immunogenicity. The vaccine can be administered inconjunction with an adjuvant, various of which are known to thoseskilled in the art. After initial immunization with the vaccine, abooster can be provided. The vaccines are administered by conventionalmethods, in dosages which are sufficient to elicit an immunologicalresponse, which can be easily determined by those skilled in the art.

Efficacy of the peptide in the context of prevention is judged based onthe following criteria: frequency of peptide reactive T cells determinedby limiting dilution, proliferation response of peptide reactive T celllines and clones, cytokine profiles of T cell lines and clones to thedesired peptide established from subjects. Efficacy is established bydecrease in frequency of reactive cells, a reduction in thymidineincorporation with altered peptide compared to native, and a reductionin TNF and IFN-α. Clinical measurements include the relapse rate in oneand two year intervals, on a Kaplan-Meier curve, a delay in sustainedcancer stage progression reduction in the number and size of tumorsincluding a change in area and volume of T2 images on MRI, and thenumber and volume of lesions determined by gadolinium enhanced images.

Peptides, variants and fragments thereof, of the present invention maybe administered either alone, or as a pharmaceutical composition.Briefly, pharmaceutical compositions of the present invention maycomprise one or more of the peptides, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like, carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide)and preservatives. In addition, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as, for example, cytokines like β-interferon.

Compositions of the present invention may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments of the invention, thecompositions described herein may be administered as part of a sustainedrelease implant. Within yet other embodiments, compositions of thepresent invention may be formulized as a lyophilizate, utilizingappropriate excipients which provide stability as a lyophilizate, andsubsequent to rehydration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the subject, and the type and severity ofthe subject's disease. Within particularly preferred embodiments of theinvention, the peptides, variants, or fragments thereof, orpharmaceutical compositions described herein may be administered at adosage ranging from about 5 to 50 mg/kg, although appropriate dosagesmay be determined by clinical trials. Dosages of peptide analogue willbe approximately 5-50 mg/kg, but are determined more accuratelyfollowing trials. Subjects may be monitored for therapeuticeffectiveness by MRI, and signs of clinical exacerbation, as describedabove.

MRI can be used to measure active lesions using gadolinium-DTPA-enhancedimaging (McDonald et al. Ann. Neurol. 36:14, 1994) or the location andextent of lesions using T₂-weighted techniques. Briefly, baseline MRIsare obtained. The same imaging plane and subject position are used foreach subsequent study. Positioning and imaging sequences are chosen tomaximize lesion detection and facilitate lesion tracing. The samepositioning and imaging sequences are used on subsequent studies. Thepresence, location and extent of MS lesions are determined byradiologists. Areas of lesions are outlined and summed slice by slicefor total lesion area. Three analyses may be done: evidence of newlesions, rate of appearance of active lesions, percentage change inlesion area (Paty et al., Neurology 43:665, 1993). Improvement due totherapy is established when there is a statistically significantimprovement in an subject subject compared to baseline or in a treatedgroup versus a placebo group.

In another aspect of the invention, any tumor antigen polypeptide can beadministered to an subject diagnosed as suffering from or susceptible tocancers. The polypeptides corresponding to identified tumor antigens canbe used to stimulate the cells of the immune system to recognize andlyse tumor cells expressing tumor antigens, such as for example, CEA,p53, K-ras, and the like.

While various procedures involving the use of antibodies have beenapplied in the treatment of tumors, few if any successful attempts usingactivated cytotoxic T-cells have been recorded. Theoretically, cytotoxicT-cells would be the preferable means of treating tumors. However, noprocedures have been available to specifically activate cytotoxicT-cells. In contrast to antibodies, the T-cell receptors on the surfaceof CD8 cells cannot recognize foreign antigens directly. Antigen mustfirst be presented to the T cell receptor, such as a dendritic cell.

The presentation of antigen to CD8 T-cells is accomplished by majorhistocompatibility complex (MHC) molecules of the Class I type. Themajor histocompatibility complex (MHC) refers to a large genetic locusencoding an extensive family of glycoproteins which play an importantrole in the immune response. The MHC genes, which are also referred toas the HLA (human leukocyte antigen) complex, are located on chromosome6 in humans. The molecules encoded by MHC genes are present on cellsurfaces and are largely responsible for recognition of tissuetransplants as “non-self”. Thus, membrane-bound MHC molecules areintimately involved in recognition of antigens by T-cells.

MHC products are grouped into three major classes, referred to as I, II,and III. T-cells that serve mainly as helper cells express CD4 andprimarily interact with Class II molecules, whereas CD8-expressingcells, which mostly represent cytotoxic effector cells, interact withClass I molecules.

Class I molecules are membrane glycoproteins with the ability to bindpeptides derived primarily from intracellular degradation of endogenousproteins. Complexes of MHC molecules with peptides derived from viral,bacterial and other foreign proteins comprise the ligand that triggersthe antigen responsiveness of T-cells. In contrast, complexes of MHCmolecules with peptides derived from normal cellular products play arole in “teaching” the T-cells to tolerate self-peptides, in the thymus.Class I molecules do not present entire, intact antigens; rather, theypresent peptide fragments thereof, “loaded” onto their “peptide bindinggroove”.

As will be recognized by those in the art, the term “host compatible” or“autologous” cells means cells that are of the same or similar haplotypeas that of the subject or “host” to which the cells are administered.

The presentation of Class I MHC molecules bound to peptide alone hasgenerally been ineffective in activating CD8 cells. In nature, the CD8cells are activated by antigen-presenting cells, such as, for example,dendritic cells, which present not only a peptide-bound Class I MHCmolecule, but also a costimulatory molecule. Such costimulatorymolecules include B7 which is now recognized to be two subgroupsdesignated as B7.1 and B7.2, ICAM-1 and LFA-3. It has also been foundthat cell adhesion molecules such as integrins assist in this process.

Dendritic cells are antigen-presenting cells that are found in alltissues and organs, including the blood. Specifically, dendritic cellspresent antigens for T lymphocytes, i.e., they process and presentantigens, and stimulate responses from naive and memory T cells. Inaddition to their role in antigen presentation, dendritic cells directlycommunicate with non-lymph tissue and survey non-lymph for an injurysignal (e.g., ischemia, infection, or inflammation) or tumor growth.Once signaled, dendritic cells initiate the immune response by releasingIL-1 which triggers lymphocytes and monocytes. When the CD8 T-cellinteracts with an antigen-presenting cell, such as a dendritic cell,having the peptide bound by a Class I MHC and costimulatory molecule,the CD8 T-cell is activated to proliferate and becomes an effectorT-cell. See, generally, Janeway and Travers, Immunobiology, published byCurrent Biology Limited, London (1994), incorporated by reference.

Accordingly, what is needed and which the present invention provides, isa means to activate T-cells so that they proliferate, become cytotoxicfor cells expressing the desired antigen, such as for example, MUC-1,and maintain memory cells specific for the administered antigen. Thus,the immune system is primed against various tumor epitopes so ifspontaneous tumors arise, a pool of primed immune cells exist whichbecome activated to recognize and kill the tumor cells.

Preferably, the epitopes presented to the immune system comprise agonistepitopes as described herein. Agonist polypeptides preferably comprisean amino acid sequence which is at least about 60% identical to theamino acid sequence of SEQ ID NO: 1 through 19, fragments or variantsthereof, more preferably, the agonist polypeptide comprises an aminoacid sequence which is at least about 80% identical to the amino acidsequence of SEQ ID NO: 1 through 19. more preferably, the agonistpolypeptide comprises an amino acid sequence which is at least about90%, 95%, or 99.9% identical to the amino acid sequence of SEQ ID NO: 1through 19.

A review of the biology of memory T cells may be found in Dutton et al.(1998) Ann. Rev Immunol 16:201-23. Memory cells express a differentpattern of cell surface markers, and they respond in several ways thatare functionally different from those of naive cells. Human memory cellsare CD45RA⁻, CD45RO⁺. In contrast to naïve cells, memory cells secrete afull range of T cell cytokines.

Chemokines and cytokines also play a powerful role in the development ofan immune response. The role of chemokines in leukocyte trafficking isreviewed by Baggiolini (1998) Nature 392:565-8, in which it is suggestedthat migration responses in the complicated trafficking of lymphocytesof different types and degrees of activation will be mediated bychemokines. The use of small molecules to block chemokines is reviewedby Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.

The role of various specific chemokines in lymphocyte homing has beenpreviously described. For example, Campbell et al. (1998) Science,showed that SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2),and MIP-3beta (also called ELC or Exodus-3) induced adhesion of mostcirculating lymphocytes, including most CD4⁺ T cells; and MIP-3alpha(also called LARC or Exodus-1) triggered adhesion of memory, but notnaïve, CD4⁺ T cells. Tangemann et al. (1998) J. Immunol. 161:6330-7disclose the role of secondary lymphoid-tissue chemokine (SLC), a highendothelial venule (HEV)-associated chemokine, with the homing oflymphocytes to secondary lymphoid organs. Campbell et al. (1998) J. CellBiol 141(4):1053-9 describe the receptor for SLC as CCR7, and that itsligand, SLC, can trigger rapid integrin-dependent arrest of lymphocytesrolling under physiological shear.

Mature B cells can be measured in immunoassays, for example, by cellsurface antigens including CD19 and CD20 with monoclonal antibodieslabeled with fluorochromes or enzymes may be used to these antigens. Bcells that have differentiated into plasma cells can be enumerated bystaining for intracellular immunoglobulins by direct immunofluorescencein fixed smears of cultured cells.

Several different ways, to assess maturity and cell differentiation, areavailable. For example, one such method is by measuring cell phenotypes.The phenotypes of immune cells and any phenotypic changes can beevaluated by flow cytometry after immunofluorescent staining usingmonoclonal antibodies that will bind membrane proteins characteristic ofvarious immune cell types.

A second means of assessing cell differentiation is by measuring cellfunction. This may be done biochemically, by measuring the expression ofenzymes, mRNA's, genes, proteins, or other metabolites within the cell,or secreted from the cell. Bioassays may also be used to measurefunctional cell differentiation or measure specific antibody productiondirected at a subject's tumor, tumor cell lines or cells from freshtumors.

Immune cells express a variety of cell surface molecules which can bedetected with either monoclonal antibodies or polyclonal antisera.Immune cells that have undergone differentiation or activation can alsobe enumerated by staining for the presence of characteristic cellsurface proteins by direct immunofluorescence in fixed smears ofcultured cells.

In vitro T cell cytotoxic assays are well known to those skilled in theart. A preferred method is to measure cytotoxicity in a 5 hr ⁵¹Sodiumchromate (⁵¹Cr) release assay. In particular, a 20 hr ⁵¹Cr-release assayis preferred. Tumor cells, also referred to herein as “target cells” areplated in flat-bottomed microtiter plates and incubated at 37° C.overnight. The targets are washed and labeled the next day with ⁵¹Cr at37° C. ⁵¹Cr is taken up by the target cells, either by endocytosis orpinocytosis, and is retained in the cytoplasm. The wells containingtumor cells are washed, and then armed or unarmed ATC, referred to as“effector cells” are plated at different E:T ratios and incubatedovernight at 37° C. Cytolysis is a measure of the ⁵¹Cr released from thetarget cells into the supernatant due to destruction of the target cellsby the effector cells. The microtiter plates are centrifuged at 1000 rpmfor 10 minutes and an aliquot of about 50 al to about 100 μl is removedand the level of radioactivity is measured the next day by a gammacounter and the percent specific lysis calculated.

Percent specific lysis is measured by using the formula:

(⁵¹Cr released from the target cells)−(spontaneous ⁵¹Cr released fromthe target cells)/(maximum ⁵¹Cr released from the targetcells)−(spontaneous ⁵¹Cr released from the target cells)×100

The spontaneous ⁵¹Cr released from the target cells is measured withtumor cells to which no effector cells have been added. Maximum ⁵¹Crreleased from the target cells is obtained by adding, for example, 1MHCl and represents the total amount of ⁵¹Cr present in the cytoplasm ofthe target cell.

Other means of assaying for T lymphocyte activity is by the mixedlymphocyte reaction described in the examples which follow. Othercytotoxicity assays such as the labeling of target cells with tritiatedthymidine (³H-TdR) may also be used. ³H-TdR is taken up by target cellsinto the nucleus of the cell. Release of ³H-TdR is a measure of celldeath by DNA fragmentation. The assay is conducted as above except theincubation period is at least about 48 hours and 50 μA to about 100 μlof the supernatant is measured by a beta-counter in the presence of atleast about 1 ml of scintillation fluid. Calculation of percent specificlysis is performed using the above formula.

In a preferred embodiment the polypeptide is expressed at least at ahigher level in a subject with cancer as compared to expression levelsin normal subjects, preferably the polypeptide is expressed at leastabout 5 to about 10 fold higher in a subject with cancer as compared toexpression in a normal subject. Preferably the cancer is a MUC-1⁺ cancerand the subject sample is obtained from a mammalian subject, including aprimate such as a human subject.

In another preferred embodiment, the invention provides for a method fortreating a subject suffering from or susceptible to a MUC-1 tumorcomprising isolating dendritic cells from a subject suffering fromcancer; and, treating the dendritic cells with one or more of thepolypeptides identified by SEQ ID NO: 1 through 19; fragments andvariants thereof. Preferably, the treated dendritic cells areadministered to the subject.

In yet another preferred embodiment, autologous dendritic cells can beisolated from a subject, transduced with the vectors described in detailherein, cultured, and re-infused into the subject.

An “isolated” or “purified” population of cells is substantially free ofcells and materials with which it is associated in nature. Bysubstantially free or substantially purified APCs is meant at least 50%of the population are APCs, preferably at least 70%, more preferably atleast 80%, and even more preferably at least 90% free of non-APCs cellswith which they are associated in nature.

Dendritic cells of different maturation stages can be isolated based onthe cell surface expression markers. For example, mature dendritic cellsare less able to capture new proteins for presentation but are muchbetter at stimulating resting T cells to grow and differentiate. Thus,mature dendritic cells can be of importance. Mature dendritic cells canbe identified by their change in morphology; by their nonadherence; andby the presence of various markers. Such markers include, but are notlimited to, cell surface markers such as B7.2, CD40, CD11c⁺, and MHCclass II. Alternatively, maturation can be identified by observing ormeasuring the production of pro-inflammatory cytokines. Dendritic cellscan be collected and analyzed using typical cytofluorography and cellsorting techniques and devices, such as a fluorescence-activated cellsorter (FACS). Antibodies specific to cell surface antigens of differentstages of dendritic cell maturation are commercially available.

The amount of dendritic cells administered to the subject will also varydepending on the condition of the subject and should be determined viaconsideration of all appropriate factors by the practitioner.Preferably, however, about 1×10⁶ to about 1×10¹², more preferably about1×10⁸ to about 1×10¹¹, and even more preferably, about 1×10⁹ to about1×10¹⁰ dendritic cells are utilized for adult humans. These amounts willvary depending on the age, weight, size, condition, sex of the subject,the type of tumor to be treated, the route of administration, whetherthe treatment is regional or systemic, and other factors. Those skilledin the art should be readily able to derive appropriate dosages andschedules of administration to suit the specific circumstance and needsof the subject.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8 cells via intravenous infusion isappropriate. Any toxicity, from donor cell infusion, observed in apregnant female will result in immediate cessation of any furtherinfusions. Toxicity is measured according to the National CancerInstitute (NCl) scale.

Toxicity Grading—The NCI Common Toxicity Scale.

-   -   If Grade I-II toxicities occur, the subject may continue with        the infusion schedule.    -   If Grade III toxicity occurs, the “drug” will be held until the        toxicity decreases to Grade I or II, then the infusion will be        restarted. If Grade III or IV toxicity occurs after the restart,        the “drug” infusions will be stopped.    -   If Grade IV toxicity occurs, the subject is scored as having        Grade IV toxicity and the next infusion is reduced to the        previous dose. If the previous dose causes Grade IV toxicity,        then the “drug” will be stopped.    -   If Grade IV toxicity occurs in 1 of 3 subjects at a specific        dose level, an additional 3 subjects must be entered at that        cell-dose level for a total of 6 subjects at that dose level. If        2 of 6 subjects at a cell-dose level develop Grade IV toxicity,        this dose is defined as the maximum tolerated dose (MTD). The        next 3 subjects will be given 66% (two-thirds) of the previous        cell-dose level. For the purposes of evaluation for        dose-escalation, each subject at the same dose level should        received at least 4 of 6 infusions.

Large quantities of antigen-presenting dendritic cells can be generatedex vivo as described in U.S. Pat. No. 6,497,876, which is incorporatedherein, in its entirety. Following collection of an subject's CD34⁺hematopoietic progenitors and stem cells, cytokines such asgranulocyte-macrophage colony stimulating factor (GM-CSF) and flt-3ligand (flt3-L) can be used to expand the cells in vitro and to drivethem to differentiate into cells of the dendritic cell lineage.Cytokines can also be used to increase the numbers of CD34⁺ cells incirculation prior to collection. The resulting dendritic cells areexposed to an antigen one wishes to elicit an immune response against,and allowed to process the antigen (this procedure is sometimes referredto in the art as “antigen-pulsing”). The antigen-pulsed (orantigen-expressing) dendritic cells are then activated with a CD40binding protein, and subsequently administered to the subject.

Dendritic cells comprise a heterogeneous cell population withdistinctive morphology and a widespread tissue distribution. Thedendritic cell system and its role in immunity is reviewed by Steinman,R. M., Annu. Rev. Immunol., 9:271-296 (1991), incorporated herein byreference. The cell surface of dendritic cells is unusual, withcharacteristic veil-like projections, and is characterized by having thecell surface markers CD1a⁺, CD4⁺, CD86⁺, or HLA-DR⁺. Dendritic cellshave a high capacity for sensitizing MHC-restricted T cells and are veryeffective at presenting antigens to T cells in situ, both self-antigensduring T cell development and tolerance and foreign antigens duringimmunity.

Because of their effectiveness at antigen presentation, autologousdendritic cells preferably are used ex vivo as alloantigen adjuvants(see, for example, Romani, et al., J. Exp. Med., 180:83 (1994). The useof dendritic cells as immunostimulatory agents has been limited due tothe low frequency of dendritic cells in peripheral blood, the limitedaccessibility of lymphoid organs and the dendritic cells' terminal stateof differentiation. Dendritic cells originate from CD34⁺ bone marrow orperipheral blood progenitors and peripheral blood mononuclear cells, andthe proliferation and maturation of dendritic cells can be enhanced bythe cytokines GM-CSF sargramostim, Leukine™ (Immunex Corporation,Seattle, Wash.), TNF-α,α-kit ligand (also known as stem cell factor(SCF), steel factor (SF), or mast cell growth factor (MGF)) andinterleukin-4. Recently, flt3-L has been found to stimulate thegeneration of large numbers of functionally mature dendritic cells, bothin vivo and in vitro.

Ex Vivo Culture of Dendritic Cells

A procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference. Other suitable methods are known in the art. Briefly, ex vivoculture and expansion comprises: (1) collecting CD34⁺ hematopoietic stemand progenitor cells from a subject from peripheral blood harvest orbone marrow explants; and (2) expanding such cells ex vivo. In additionto the cellular growth factors described in U.S. Pat. No. 5,199,942,other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used.

Stem or progenitor cells having the CD34 marker constitute only about 1%to 3% of the mononuclear cells in the bone marrow. The amount of CD34⁺stem or progenitor cells in the peripheral blood is approximately 10- to100-fold less than in bone marrow. Cytokines such as flt3-L may be usedto increase or mobilize the numbers of dendritic cells in vivo.Increasing the quantity of a subject's dendritic cells may facilitateantigen presentation to T cells for antigen(s) that already existswithin the subject, such as a tumor antigen, or a bacterial or viralantigen. Alternatively, cytokines may be administered prior to,concurrently with or subsequent to administration of an antigen to ansubject for immunization purposes.

Peripheral blood cells are collected using apheresis procedures known inthe art. See, for example, Bishop et al., Blood, vol. 83, No. 2, pp.610-616 (1994). Briefly, peripheral blood progenitor cells (PBPC) andperipheral blood stein cells (PBSC) are collected using conventionaldevices, for example, a Haemonetics Model V50 apheresis device(Haemonetics, Braintree, Mass.). Four-hour collections are performedtypically no more than five times weekly until approximately 6.5×10⁸mononuclear cells (MNC)/kg are collected. The cells are suspended instandard media and then centrifuged to remove red blood cells andneutrophils. Cells located at the interface between the two phases (thebuffy coat) are withdrawn and resuspended in HBSS. The suspended cellsare predominantly mononuclear and a substantial portion of the cellmixture are early stem cells.

A variety of cell selection techniques are known for identifying andseparating CD34⁺ hematopoietic stem or progenitor cells from apopulation of cells. For example, monoclonal antibodies (or otherspecific cell binding proteins) can be used to bind to a marker proteinor surface antigen protein found on stem or progenitor cells. Severalsuch markers or cell surface antigens for hematopoietic stem cells(i.e., flt-3, CD34, My-10, and Thy-1) are known in the art, as arespecific binding proteins.

In one method, antibodies or binding proteins are fixed to a surface,for example, glass beads or flask, magnetic beads, or a suitablechromatography resin, and contacted with the population of cells. Thestem cells are then bound to the bead matrix. Alternatively, the bindingproteins can be incubated with the cell mixture and the resultingcombination contacted with a surface having an affinity for theantibody-cell complex. Undesired cells and cell matter are removedproviding a relatively pure population of stem cells. The specific cellbinding proteins can also be labeled with a fluorescent label, e.g.,chromophore or fluorophore, and the labeled cells separated by sorting.Preferably, isolation is accomplished by an immunoaffinity column.

Immunoaffinity columns can take any form, but usually comprise a packedbed reactor. The packed bed in these bioreactors is preferably made of aporous material having a substantially uniform coating of a substrate.The porous material, which provides a high surface area-to-volume ratio,allows for the cell mixture to flow over a large contact area while notimpeding the flow of cells out of the bed. The substrate should, eitherby its own properties, or by the addition of a chemical moiety, displayhigh-affinity for a moiety found on the cell-binding protein. Typicalsubstrates include avidin and streptavidin, while other conventionalsubstrates can be used.

In one useful method, monoclonal antibodies that recognize a cellsurface antigen on the cells to be separated are typically furthermodified to present a biotin moiety. The affinity of biotin for avidinthereby removably secures the monoclonal antibody to the surface of apacked bed (see Berenson, et al., J. Immunol. Meth., 91:11, 1986). Thepacked bed is washed to remove unbound material, and target cells arereleased using conventional methods. Immunoaffinity columns of the typedescribed above that utilize biotinylated anti-CD34 monoclonalantibodies secured to an avidin-coated packed bed are described forexample, in WO 93/08268.

An alternative means of selecting the quiescent stem cells is to inducecell death in the dividing, more lineage-committed, cell types using anantimetabolite such as 5-fluorouracil (5-FU) or an alkylating, agentsuch as 4-hydroxycyclophosphamide (4-HC). The non-quiescent cells arestimulated to proliferate and differentiate by the addition of growthfactors that have little or no effect on the stem cells, causing thenon-stem cells to proliferate and differentiate and making them morevulnerable to the cytotoxic effects of 5-FU or 4-HC. See Berardi et al.,Science, 267:104 (1995), which is incorporated herein by reference.

Isolated stem cells can be frozen in a controlled rate freezer (e.g.,Cryo-Med, Mt. Clemens, Mich.), then stored in the vapor phase of liquidnitrogen using dimethylsulfoxide as a cryoprotectant. A variety ofgrowth and culture media can be used for the growth and culture ofdendritic cells (fresh or frozen), including serum-depleted orserum-based media. Useful growth media include RPMI, TC 199, Iscovesmodified Dulbecco's medium (Iscove, et al., F. J. Exp. Med., 147:923(1978)), DMEM, Fischer's, alpha medium, NCTC, F-10, Leibovitz's L-15,MEM and McCoy's. Particular nutrients present in the media include serumalbumin, transferrin, lipids, cholesterol, a reducing agent such as2-mercaptoethanol or monothioglycerol, pyruvate, butyrate, and aglucocorticoid such as hydrocortisone 2-hemisuccinate. Moreparticularly, the standard media includes an energy source, vitamins orother cell-supporting organic compounds, a buffer such as HEPES, orTris, that acts to stabilize the pH of the media, and various inorganicsalts. A variety of serum-free cellular growth media is described in WO95/00632, which is incorporated herein by reference. The collected CD34⁺cells are cultured with suitable cytokines, for example, as describedherein. CD34⁺ cells then are allowed to differentiate and commit tocells of the dendritic lineage. These cells are then further purified byflow cytometry or similar means, using markers characteristic ofdendritic cells, such as CD1a, HLA DR, CD80 and/or CD86. The cultureddendritic cells are exposed to an antigen, for example, an allogeneicclass I HLA molecule, allowed to process the antigen, and then culturedwith an amount of a CD40 binding protein to activate the dendritic cell.Alternatively, the dendritic cells are transfected with a gene encodingan allogeneic HLA class 1 molecule or immune related receptors, and thencultured with an amount of a CD40 binding protein to activate theantigen-presenting dendritic cells.

The activated, antigen-carrying dendritic cells are them administered toan subject in order to stimulate an antigen-specific immune response.The dendritic cells can be administered prior to, concurrently with, orsubsequent to, antigen administration. Alternatively, T cells may becollected from the subject and exposed to the activated,antigen-carrying dendritic cells in vitro to stimulate antigen-specificT cells, which are administered to the subject.

Useful Cytokines

Various cytokines will be useful in the ex vivo culture of dendriticcells. Flt3-L refers to a genus of polypeptides that are described in EP0627487 A2 and in WO 94/28391, both incorporated herein by reference. Ahuman flt3-L cDNA was deposited with the American Type CultureCollection, Rockville, Md., USA (ATCC) on Aug. 6, 1993 and assignedaccession number ATCC 69382. IL-3 refers to a genus of interleukin-3polypeptides as described in U.S. Pat. No. 5,108,910, incorporatedherein by reference. A DNA sequence encoding human IL-3 protein suitablefor use in the invention is publicly available from the American TypeCulture Collection (ATCC) under accession number ATCC 67747. c-kitligand is also referred to as Mast Cell Growth Factor (MGF), SteelFactor or Stern Cell Factor (SCF), and is described in EP 423,980, whichis incorporated herein by reference. Other useful cytokines includeInterleukin-4 (IL-4; Mosley et al., Cell 59:335 (1989), Idzerda et al.,J. Exp. Med. 171:861 (1990) and Galizzi et al., Intl. Immunol. 2:669(1990), each of which is incorporated herein by reference) andgranulocyte-macrophage colony stimulating factor (GM-CSF; described inU.S. Pat. Nos. 5,108,910, and 5,229,496 each of which is incorporatedherein by reference). Commercially available GM-CSF (sargramostim,Leukine™) is obtainable from Immunex Corp., Seattle, Wash.). Moreover,GM-CSF/IL-3 fusion proteins (i.e., a C-terminal to N-terminal fusion ofGM-CSF and IL-3) will also be useful in ex vivo culture of dendriticcells. Such fusion proteins are known and are described in U.S. Pat.Nos. 5,199,942, 5,108,910 and 5,073,627, each of which is incorporatedherein by reference. A preferred fusion protein is PIXY321 as describedin U.S. Pat. No. 5,199,942.

Useful cytokines act by binding a receptor present on the surface of adendritic cell and transducing a signal. Moreover, additional bindingproteins can be prepared as described herein for CD40 binding proteins,that bind appropriate cytokine receptors and transduce a signal to adendritic cell. For example, WO 95/27062 describes agonistic antibodiesto Flt-3, the receptor for Flt-3L, from which various Flt-3 binding.proteins can be prepared. Additional useful cytokines includebiologically active analogs of cytokines that are useful for culturingdendritic cells. Useful cytokine analogs have an amino acid sequencethat is substantially similar to the native cytokine, and arebiologically active capable of binding to their specific receptor andtransducing a biological signal. Such analogs can be prepared and testedby methods that are known in the art.

An alternate method for preparing dendritic cells that present antigenis to transfect the dendritic cells with a gene encoding an antigen or aspecific polypeptide derived therefrom. Once the dendritic cells expressthe antigen in the context of MHC, the dendritic cells are activatedwith a CD40 binding protein, and subsequently administered to thesubject to provide a stronger and improved immune response to theantigen.

The activated antigen-presenting dendritic cells can also be used as avaccine adjuvant and can be administered prior to, concurrently with orsubsequent to antigen administration. Moreover, the dendritic cells canbe administered to the subject prior to, concurrently with or subsequentto administration of cytokines that modulate an immune response, forexample a CD40 binding protein (i.e., soluble CD40L), or a soluble CD83molecule. Additional useful cytokines include, but are not limited to,Interleukins (IL) 1, 2, 4, 5, 6, 7, 10, 12 and 15, colony stimulatingfactors (CSF) such as GM-CSF, granulocyte colony stimulating factor(G-CSF), or GM-CSF/IL-3 fusion proteins, or other cytokines such asTNF-α, or c-kit ligand. Moreover, biologically active derivatives ofthese cytokines; and combinations thereof will also be useful.

CD40 is a member of the tumor necrosis factor (TNF)/nerve growth factor(NGF) receptor family, which is defined by the presence of cysteine-richmotifs in the extracellular region (Smith et al., Science 248:1019,1990; Mallett and Barclay, Immunology Today 12:220; 1991). This familyincludes the lymphocyte antigen CD27, CD30 (an antigen found onHodgkin's lymphoma and Reed-Sternberg cells), two receptors for TNF, amurine protein referred to as 4-1BB, rat OX40 antigen, NGF receptor, andFas antigen. Human CD40 antigen (CD40) is a peptide of 277 amino acidshaving a molecular weight of 30,600 (Stamenkovic et al., EMBO J. 8:1403,1989). CD40L is believed to be important in feedback regulation of animmune response. For example, a CD40″ antigen presenting cell willpresent antigen to a T cell, which will then become activated andexpress CD40L. The CD40L will, in turn, further activate the antigenpresenting cell, increasing its efficiency at antigen presentation, andupregulating expression of Class I and Class II MHC, CD80 and CD86costimulatory molecules, as well as various cytokines (Caux et al., J.Exp. Med. 180:1263, 1994).

Purified dendritic cells are then pulsed with (exposed to) antigen, toallow them to take up the antigen in a manner suitable for presentationto other cells of the immune systems. Antigens are classically processedand presented through two pathways. Peptides derived from proteins inthe cytosolic compartment are presented in the context of Class I MHCmolecules, whereas peptides derived from proteins that are found in theendocytic pathway are presented in the context of Class II MHC. However,those of skill in the art recognize that there are exceptions; forexample, the response of CD8⁺ tumor specific T cells, which recognizeexogenous tumor antigens expressed on MHC Class I. A review ofMHC-dependent antigen processing and peptide presentation is found inGermain, R. N., Cell 76:287 (1994).

Numerous methods of pulsing dendritic cells with antigen are known;those of skill in the art regard development of suitable methods for aselected antigen as routine experimentation. In general, the antigen isadded to cultured dendritic cells under conditions promoting viabilityof the cells, and the cells are then allowed sufficient time to take upand process the antigen, and express antigen peptides on the cellsurface in association with either Class I or Class II MHC, a period ofabout 24 hours (from about 18 to about 30 hours, preferably 24 hours).Dendritic cells may also be exposed to antigen by transfecting them withDNA encoding the antigen. The DNA is expressed, and the antigen ispresumably processed via the cytosolic/Class I pathway.

The present invention provides methods of using therapeutic compositionscomprising activated, antigen-pulsed dendritic cells. The use of suchcells in conjunction with soluble cytokine receptors or cytokines, orother immunoregulatory molecules is also contemplated. The inventivecompositions are administered to stimulate an allogeneic immuneresponse, and can be given by bolus injection, continuous infusion,sustained release from implants, or other suitable technique. Typically,the cells on the will be administered in the form of a compositioncomprising the antigen-pulsed, activated dendritic cells in conjunctionwith physiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed. Neutral buffered saline or saline mixed withserum albumin are exemplary appropriate diluents.

For use in stimulating a certain type of immune response, administrationof other cytokines along with activated, antigen-pulsed dendritic cellsis also contemplated. Several useful cytokines (or peptide regulatoryfactors) are discussed in Schrader, J. W. (Mol. Immunol 28:295; 1991).Such factors include (alone or in combination) Interleukins 1, 2, 4, 5,6, 7, 10, 12 and 15; granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor; a fusion protein comprisingInterleukin-3 and granulocyte-macrophage colony stimulating factor;Interferon-γ, TNF, TGF-13, flt-3 ligand and biologically activederivatives thereof. A particularly preferred cytokine is CD40 ligand(CD40L). Other cytokines will also be useful, as described herein. DNAencoding such cytokines will also be useful in the inventive methods,for example, by transfecting the dendritic cells to express thecytokines. Administration of these immunomodulatory molecules includessimultaneous, separate or sequential administration with the cells ofthe present invention.

In another preferred embodiment, the invention provides for apolypeptide identified by any one of SEQ ID NO: 1 through 19 having asequence identity to anyone one of SEQ ID NO: 1 through 19 of at leastabout 10%, more preferably, 25%, even more preferably about 40%, 50%,60%, 70%, 80%, 90%, or 99.9%. Dendritic cells can be pulsed with any ofthese polypeptides during ex-vivo culture.

In one aspect of the invention, the polypeptide comprises SEQ ID NO: 19.Preferably, the polypeptide binds to HLA molecules with a high avidityand has a higher association constant (K_(a)) for the HLA than a nativepolypeptide and/or a lower dissociation constant (K_(d)) for the HLAthan a native polypeptide.

In another aspect of the invention, the polypeptide is derived from amucin tumor antigen, preferably, the polypeptide is derived from anon-variable number of tandem repeats region of MUC-1.

In another aspect of the invention, antigen presentation, by antigenpresenting cells of the polypeptides induces an immune response,preferably a cellular immune response. For example, the cellular immuneresponse is a cytotoxic T cell response, a T helper cell response, or aB cell immune response.

In yet another aspect, variants of the nucleic acid molecule encodingpolypeptides as identified by SEQ ID NO: 1 through 19 can be used totransduce immune cells for the detection and lysing of, for example,MUC-1 positive cancers. An “allele” or “variant” is an alternative formof a gene. Of particular utility in the invention are variants of thegenes encoding any potential MUC-1⁺ tumor cell markers identified by themethods of this invention. Variants may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orin polypeptides whose structure or function may or may not be altered.Any given natural or recombinant gene may have none, one, or manyallelic forms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The compositions and methods of the present invention also encompassvariants of the above polypeptides and nucleic acid sequences encodingsuch polypeptides. A polypeptide “variant,” as used herein, is apolypeptide that differs from the native polypeptide in substitutionsand/or modifications, such that the antigenic and/or immunogenicproperties of the polypeptide are retained. Such variants may generallybe identified by modifying one of the above polypeptide sequences andevaluating the reactivity of the modified polypeptide with antiseraand/or T-cells as described above. Nucleic acid variants may contain oneor more substitutions, deletions, insertions and/or modifications suchthat the antigenic and/or immunogenic properties of the encodedpolypeptide are retained. One preferred variant of the polypeptidesdescribed herein is a variant that contains nucleotide substitutions,deletions, insertions and/or modifications at no more than 20% of thenucleotide positions.

Preferably, but not limited to, a variant contains conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his. However, any type ofsubstitution is within the scope and embodiments of the invention.

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenic or antigenic properties, secondary structure and hydropathicnature of the polypeptide. For example, a polypeptide may be conjugatedto a signal (or leader) sequence at the N-terminal end of the proteinwhich co-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification or identification of thepolypeptide (e.g., poly-His), or to enhance binding of the polypeptideto a solid support. For example, a polypeptide may be conjugated to animmunoglobulin Fc region.

In general, nucleotide sequences encoding all or a portion of thepolypeptides described herein may be prepared using any of severaltechniques. For example, cDNA molecules encoding such polypeptides maybe cloned on the basis of the MUC-1 tumor-specific expression of thecorresponding mRNAs, using differential display PCR. This techniquecompares the amplified products from RNA template prepared from normaland MUC-1 positive tumor tissue. cDNA may be prepared by reversetranscription of RNA using a random primer, such as for example, (dT)₁₂AG primer. Following amplification of the cDNA using a random primer, aband corresponding to an amplified product specific to the tumor RNA maybe cut out from a silver stained gel and subcloned into a suitablevector, such as the adenovirus vector described in the examples whichfollow. Nucleotide sequences encoding all or a portion of the MUC-1tumor-specific polypeptides disclosed by any one of SEQ ID NOs:1 through6 and variants thereof may be amplified from cDNA prepared as describedabove using any random primers.

Alternatively, a gene encoding a polypeptide as described herein (or aportion thereof) may be amplified from human genomic DNA, or from tumorcell cDNA, via polymerase chain reaction.

In an embodiment of the invention the presence of the one or morenucleic acid molecules is correlated to a sample of a normal subject.The sample is preferably obtained from a mammal suspected of having aproliferative cell growth disorder, in particular, a MUC-1⁺ cancer.

Percent identity and similarity between two sequences (nucleic acid orpolypeptide) can be determined using a mathematical algorithm (see,e.g., Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

In another preferred embodiment, MUC-1 peptide fragments and derivativesof the invention are of a sufficient length such that they activate theimmune system resulting in the lysing of cancer cells, such as, forexample cells expressing MUC-1. MUC-1 nucleic acid molecules, fragmentsand derivatives encoding for any one of the polypeptides identified bySEQ ID NO: 1 through 19, thus preferably comprise at least about 90%nucleotides as compared to the sequence identified by any one of SEQ IDNO: 1 through 19, usually at least about 80% nucleotides as compared tothe sequence identified by any one of SEQ ID NO: 1 through 19, moreusually at least about 70% nucleotides as compared to the sequenceidentified by anyone of SEQ ID NO: 1 through 19, even more typically atleast about 40% or 50% nucleotides.

To determine the percent identity of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, or 90% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position (as used herein nucleic acid “identity” is equivalent tonucleic acid “sequence identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at online through the Genetics ComputerGroup), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70,or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred,non-limiting example of parameters to be used in conjunction with theGAP program include a Blosum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of Meyers andMiller (Comput. Appl. Biosci. 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The treatment of neoplastic disease or neoplastic cells, refers to anamount of the vectors and/or peptides, described throughout thespecification and in the Examples which follow, capable of invoking oneor more of the following effects: (1) inhibition, to some extent, oftumor growth, including, (i) slowing down and (ii) complete growtharrest; (2) reduction in the number of tumor cells; (3) maintainingtumor size; (4) reduction in tumor size; (5) inhibition, including (i)reduction, (ii) slowing down or (iii) complete prevention, of tumor cellinfiltration into peripheral organs; (6) inhibition, including (i)reduction, (ii) slowing down or (iii) complete prevention, ofmetastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion or (v) reducing, slowing or preventing metastasis; and/or (8)relief, to some extent, of one or more symptoms associated with thedisorder.

Thus in one aspect of the invention any variant, fragment, mutant can beused to transduce immune cells, such as for example dendritic cells, forthe treatment of an subject suffering from, or, prophylactically to ansubject susceptible to cancer. As discussed above, a preferred use ofnucleic acid sequences identified in the present invention, is for thegeneration of treatments that lyse for example, MUC-1 cancer cells. Thenucleic acid molecules can be expressed by a vector containing a DNAsegment encoding the wild-type, alleles, variants, mutations orfragments of the genes. Mutations and alleles of the nucleic acidmolecules are also preferably used in the construction of a vector foruse in treatment. The vector comprising the desired nucleic acidsequence for conferring resistance to, for example, MUC-1 positivecancer, preferably has at least one such nucleic acid sequence.Alternatively, the vector may be comprised of more than one such nucleicacid sequence, or combinations of allelic variants. The vector can alsobe comprised of cassettes of different allelic variants or wild typenucleic acid molecules.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Introducing the genes, fragments or alleles thereof, into an subject caninclude use of vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/04701, which has a targeting moiety (e.g. a ligand toa cellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

In another preferred embodiment, cells are isolated and purified cellfrom a sample, subject or donor subject and are used in functionalassays to determine any properties of the cells. Depending on theisolated and purified cellular population, appropriate functional assaysknown in the art can be conducted. For example, if the population ofcells are T cells specific for a desired antigen such as a tumorantigen, cytotoxic T cell assays, T cell proliferation assays, cytokineprofiles, determination of surface antigens for T cell maturity ormemory T cells, etc., can be carried out.

Isolation of cells useful in the present invention are well known in theart. For example, peripheral blood mononuclear cells (PBMCs) can beobtained from a subject and isolated by density gradient centrifugation,e.g., with Ficoll/Hypaque. Specific cell populations can be depleted orenriched using standard methods. For example, monocytes/macrophages canbe isolated by adherence on plastic. T cells or B cells can be enrichedor depleted, for example, by positive and/or negative selection usingantibodies to T cell or B cell surface markers, for example byincubating cells with a specific primary monoclonal antibody (mAb),followed by isolation of cells that bind the mAb using magnetic beadscoated with a secondary antibody that binds the primary mAb. Peripheralblood or bone marrow derived hematopoietic stem cells can be isolated bysimilar techniques using stem cell-specific mAbs (e.g., anti-CD34 mAbs).Specific cell populations can also be isolated by fluorescence activatedcell sorting according to standard methods. Monoclonal antibodies tocell-specific surface markers known in the art and many are commerciallyavailable.

If desired, a large proportion of terminally differentiated cells may beremoved by initially using a “relatively crude” separation. For example,magnetic bead separations may be used initially to remove large numbersof lineage committed cells. Desirably, at least about 80%, usually atleast 70% of the total hematopoietic cells can be removed.

Procedures for separation may include but are not limited to, magneticseparation, using antibody-coated magnetic beads, affinitychromatography, cytotoxic agents joined to a monoclonal antibody or usedin conjunction with a monoclonal antibody, including but not limited to,complement and cytotoxins, and “panning” with antibody attached to asolid matrix, e.g., plate, elutriation or any other convenienttechnique.

Procedures for screening can include methods of screening for moleculesto generate an immune response to a MUC-1 tumor antigen. The methods mayinclude:

altering a nucleic acid encoding a portion of the non-variable number oftandem repeats of MUC-1;

-   -   expressing the altered nucleic acid to produce a molecule;    -   contacting a dendritic cell with the molecule; and    -   contacting a T-cell with the dendritic cell,

wherein a modulation of the IFN-γ production of the T-cell indicatesthat the molecule may generate an immune response.

Techniques providing accurate separation include but are not limited to,flow cytometry, which can have varying degrees of sophistication, e.g.,a plurality of color channels, low angle and obtuse light scatteringdetecting channels, impedance channels, etc.

The peptide disclosed herein may be encoded by the correspondingsequences listed herein, but may also be encoded by degenerat codons,including:

Amino acid Code A GCT, GCC, GCA, GCG R CGT, CGC, CGA, CGG, AGA, AGG NAAT, AAC D GAT, GAC C TGT, TGC G GGT, GGC, GGA, GGG Q CAA, CAG EGAA, GAG H CAT, CAC I ATC, ATT, ATA L TTA, TTG, CTT, CTC, CTA, CTG KAAA, AAG M ATG F TTT, TTC P CCT, CCC, CCA, CCG STCT, TCC, TCA, TCG, AGT, AGC T ACT, ACC, ACA, ACG W TGG Y TAT, TAC VGTT, GTC, GTA, GTG

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention. The following non-limiting examples are illustrative of theinvention.

EXAMPLES Example 1 Materials and Methods

Cell Cultures.

The human breast adenocarcinoma cell line MCF-7 (HLA-A2 positive andMUC-1 positive), and SK-MeI-24 (HLA-A2 positive, MUC-1 negative) werepurchased from American Type Culture Collection (Manassas, Va.). Thecultures were free of mycoplasma and were maintained in complete medium[RPMI 1640 (Invitrogen Life Technologies, Carlsbad, Calif.) supplementedwith 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin,and 100 μg/ml streptomycin (Invitrogen Life Technologies, Inc.)]. TheC1R cell line is a human plasma leukemia cell line that does not expressendogenous HLA-A or B antigens. C1R-A2 cells are C1R cells that expressa transfected genomic clone of HLA-A2.1. These cells were obtained fromDr. William E. Biddison (National Institute of Neurological Disordersand Stroke, NIH, Bethesda, Md.). The 174CEM-T2 cell line (T2) transportdeletion mutant was provided by Dr. Peter Cresswell (Yale UniversitySchool of Medicine, New Haven, Conn.). C1R-A2 cells and T2 cells weremycoplasma free and were maintained in RPMI 1640 complete medium and inIscove's modified Dulbecco's complete medium (Invitrogen LifeTechnologies), respectively.

Peptides.

The amino acid sequence of MUC-1 was scanned for matches to consensusmotifs for HLA-A2 binding peptides. The computer algorithm from theBioInformatics and Molecule Analysis Section of NIH (BIMAS) that wasdeveloped by Parker K. C. et al., (J. Immunol., 152: 163-175, 1994) wasused, which ranks potential MHC binding peptides according to thepredictive one-half-time dissociation of peptide/MHC complexes. TheHLA-A2 allele was chosen because they are the most commonly expressedclass I allele. Ten-mer peptides from the non-variable number of tandemrepeat sequence were synthesized if they conformed to the respectiveconsensus motif. A panel of 10-mer MUC-1 peptides (Table 1) andanalogues with single amino acid substitution to positions P1 to P10 ofP-92 MUC-1 peptide (see FIG. 1) were made by American Peptide Company(Sunnyvale, Calif.) with purity>90%. In addition, a CEA peptide CAP1-6D(28)>96% pure was made by American Peptide Company (Sunnyvale, Calif.).

Flow Cytometric Analysis.

(i) Single-Color Flow Cytometric Analysis.

The method for single-color flow cytometric analysis has been describedpreviously (Gaudagni F. et al. Cancer Res.: 50: 6248-6255, 1990.).Briefly, cells were washed three times with cold Ca²⁺ and Mg²⁺-free DPBSand then stained for 1 h at 4° C. using 1 μg of the mAb against HLA-A2(A2, 28, One Lambda, Inc., Canoga Park, Calif.), CD3, CD4, CD8, and CD56(BD Biosciences, San Jose, Calif.). Mineral oil plasmacytoma-104E(Cappel/Organon Teknika Corp., West Chester, Pa.) was used as an isotypecontrol. The cells were then washed three times and incubated with a1:100 dilution of fluorescein isothiocyanate (FITC)-labeled goatanti-mouse immunoglobulin (IgG) (Kirkegaard and Perry Laboratories,Gaithersburg, Md.). The cells were immediately analyzed using a BectonDickinson FACScan equipped with a blue laser with an excitation of 15 nWat 488 nm. Data were gathered from 10,000 live cells, stored and used togenerate results.

(ii) Dual-Color Flow Cytometric Analysis.

The procedure for dual-color flow cytometric analysis was similar tothat for single-color analysis with the following exceptions.Anti-MHC-class II FITC/anti-CD11c PE, anti-MHC-class II FITC/anti-CD80PE, anti-CD58 FITC/anti-CD54 PE, anti-MHC class I FITC/anti-MHC class IIPE, and anti-IgG1 FITC/anti-IgG2a PE (isotype controls) were used forthe analysis of dendritic cells; >96% of the dendritic cells were CD11cand MHC class II positive.

The antibodies used for the analysis of T cell lines were anti-CD56FITC/anti-CD8 PE, anti-CD4 FITC/anti-CD8 PE andanti-CD45RO-FITC/anti-CD49d PE; >98% of the T-1191-P92 and T-1191-P-93Lcells were CD8 positive. Antibodies to CD4, CD8, CD28, CD45RO, CD56,CD49d, CD54, CD80, CD86, CD58 and CD11c were purchased from BDBiosciences. Antibodies to MHC-class I and MHC-class II were purchasedfrom Serotec, Oxford, UK. Staining was done simultaneously for 1 h,after which cells were washed three times, resuspended as above, andimmediately analyzed using a Becton Dickinson FACScan equipped with ablue laser with an excitation of 15 mW at 488 nm with the use of theCELLQuest program.

Results were expressed in % of positive cells and mean fluorescenceintensity (MFI). MFI was used to express the levels of fluorescencedetermined by measuring the average for all the cells in the gatedfluorescence dot plot. The MFI value was collected in log scale on theFACSCAN.

Peptide Binding to HLA-A2.

Binding of P-92 and P-92 analogues to HLA-A2 molecules was evaluated bybinding to T2A2 cells as demonstrated by flow cytometry. In this assay,increased stability (accumulation) of HLA-A2 molecules on the surface ofT2 cells as a consequence of peptide binding is measured by increasedbinding of antibody directed against HLA-A2 molecule. Briefly, 1×10⁶cells in serum-free Iscove's modified Dulbecco's complete medium wereincubated with peptides at a concentration of 50 μg/ml in 24-wellculture plates at 37° C. in 5% CO₂ Flow cytometry for peptide bindingwas performed using T2 cells and single-color analysis. After cells werewashed three times in DPBS, as described above, they were incubated for1 h with a 1:100 dilution of HLA-A2-specific MAb (One Lambda, Inc.) per10⁶ cells. UPC-10 (Cappel/Organon Teknika) was used as isotype control.The cells were then washed three times and incubated with 1:100 dilutionof FITC-labeled anti-mouse IgG (BD Biosciences). Analysis was conductedwith the FACScan, as described above. Cells were maintained on iceduring all cell preparation and staining.

Culture of DCs from PBMCs.

HLA-A2 normal donor PBMCs were obtained from heparinized blood. PBMCswere separated using lymphocyte separation medium gradient (OrganonTeknika, Durham, N.C.), as described previously (Boyum, A. Scand. J.Clin. Lab. Invest. 97(Suppl): 51-76, 1968.). DCs were prepared using amodification of the procedure described by Sallusto et al. (J. Exp.Med., 179: 1109-1118, 1994.). PBMCs (1.5×10⁸) were resuspended in AIM-Vmedium containing 2 mM glutamine, 50 μg/ml streptomycin, and 10 μg/mlgentamycin (Invitrogen Life Technologies, Inc.), and allowed to adhereto a T-150 flask (Corning Costar Corp., Cambridge, Mass.). After 2 h at37° C., the non-adherent cells were removed with a gentle rinse. Theadherent cells were cultured for 6-7 days in AIM-V medium containing 100ng/ml of recombinant human GM-CSF (rhGM-CSF) and 20 ng/ml of recombinanthuman IL-4 (rhIL-4). The culture medium was replenished every 3 days.

Recombinant Virus and Infection of DCs with Avipox Virus ContainingMUC-1 (rF-MUC-1/TRICOM).

Recombinant fowlpox virus was constructed as described by Jenkins S., etal., AIDS Res. Hum. Retroviruses, 7: 991-998, 1991. A plaque-purifiedisolate from the PDXVAC-TC vaccine strain of fowlpox virus was used asthe parental virus for this recombinant virus. The MUC-1, LFA-3, ICAM-1,and B7-1 sequences were inserted into the BamHI, J region of the fowlpoxvirus genome. In addition, the lacZ gene, under the control of thefowlpox C1 promoter, was included to identify and isolate recombinantviruses using a chromogenic assay for β-galactosidase. The MUC-1 genethat was inserted into fowlpox virus varies from the native MUC-1 gene.It encodes a 30-amino acid signal sequence, followed by the first 38amino acids of the mature N-terminal sequence of the MUC-1 protein, 10identical copies of the 20-amino acid repeated sequence, and theC-terminal portion of the protein. The 600 bp repeated sequence wasproduced using overlapping synthetic oligonucleotides containing codonswith numerous third-base variations in each repeat sequence withoutchanging the encoded amino acids. This was done to minimize duplicatednucleotide sequences, which are unstable in pox viruses, whilemaintaining the repeated amino acid sequences.

rF-MUC-1/TRICOM is a recombinant fowlpox virus that contains the MUC-1gene under the control of the 40K promoter, the human LFA-3 gene underthe control of the vaccinia 30K promoter, the human ICAM-1 gene underthe control of the vaccinia 13 promoter, and the human B7-1 gene underthe control of the synthetic early/late (sE/L) promoter. Dendritic cells(DCs) (1×10⁶) were incubated in 1 ml of Opti-MEM medium (LifeTechnologies, Inc.) at 37° C. with rF-MUC-1/TRICOM or control avipoxvirus vector (FP-WT). Titration experiments demonstrated that 4×10⁷plaque-forming units/ml, equal to an MOI of 40:1 for 2 h, were able toconsistently induce transgene expression in approximately 75% of theinfected DCs. The infected DCs were suspended in 10 ml of fresh, warmRPMI-1640 complete medium containing 100 ng/ml of rhGM-CSF, 20 ng/mlrhIL-4, and 20 ng/ml of TNF-α cultured for 24 h, and then subsequentlyused as APCs.

Generation of T-Cell Lines.

Modification of the protocol described by Tsang K. Y. et al., J. Natl.Cancer Inst., 87: 982-990, 1995, was used to generate MUC-1-specificCTL. To generate T-cell lines T-1191-P-93L and T-1191-P-92, autologousDCs infected with rF-MUC-1/TRICOM were used as APCs. Autologousnon-adherent cells were then added to APCs at an effector-to-APC ratioof 10:1. Cultures were then incubated for 3 days at 37° C. in ahumidified atmosphere containing 5% CO2. The cultures were thensupplemented with recombinant human IL-2 at a concentration of 20units/ml for 7 days; the IL-2 containing medium was replenished every 3days. The 3-day incubation with peptide and 7-day IL-2 supplementconstituted one IVS cycle. Primary cultures were restimulated withrF-MUC-1/TRICOM-infected autologous DCs as described above on day 11 tobegin the next IVS cycle. rF-MUC-1/TRICOM-infected autologous DCs wereused as APCs for three IVS cycles. Irradiated (23,000 rads) autologousEBV-transformed B cells were used as APCs after the third IVS cycle. Forthe restimulation with EBV-transformed B cells, peptides at aconcentration of 50 mg/ml were used to pulse the autologousEBV-transformed B cells at a ratio of effector-to-APC of 1:3 forrestimulation. Cultures were then incubated for 3 days at 37° C. in ahumidified atmosphere containing 5% CO2. After removal of the peptidecontaining medium, the cultures were then supplemented with recombinanthuman IL-2 at a concentration of 20 unit/ml for days. T cell lines fromsubjects 18 and 23 (T-18-P-92, T-18-P93L, T-23-P-92 and T-23-P93L) weregenerated by stimulation of PBMCs with autologous DCs pulsed with theP-92 or P93L peptides using the same stimulation protocol as describedabove. The markers used for the analysis and identification of DCs wereCD11c, MHC-class II, CD80, CD54, CD58 and CD83. CD3 was also used as anegative marker.

Cytotoxic Assay.

Target cells (C1R-A2 or tumor cells) were labeled with 50 μCi of¹¹¹Indium-labeled oxyquinoline (Medi-Physics Inc., Arlington, Ill.) for15 min at room temperature. Target cells (0.3×10⁴) in 100 μl ofRPMI-1640 complete medium were added to each of 96 wells inflat-bottomed assay plates (Corning Costar Corp.). Labeled C I R-A2target cells were incubated with peptides at the concentration indicatedfor 60 min at 37° C. in 5% CO₂ before adding effector cells. No peptidewas used when carcinoma cell lines were used as targets. Effector cellswere suspended in 100 μl of RPMI-1640 complete medium supplemented with10% pooled human AB serum and added to the target cells. The plates werethen incubated at 37° C. in 5% CO₂ for 4 or 16 h. Supernatant washarvested for gamma counting with the use of harvester frames (Skatron,Inc., Sterling, Va.). Determinations were carried out in triplicate, andstandard deviations were calculated. Specific lysis was calculated withthe use of the following formula (all values in cpm):

${\% \mspace{14mu} {lysis}} = {\frac{{{Observed}\mspace{14mu} {release}} - {{Spontaneous}\mspace{14mu} {release}}}{{{Total}\mspace{14mu} {release}} - {{Spontaneous}\mspace{14mu} {release}}} \times 100}$

Spontaneous release was determined from wells to which 100 μl ofRPMI-1640 complete medium was added. Total releasable radioactivity wasobtained after treatment of targets with 2.5% Triton X-100.

Detection of Cytokines.

Supernatants of T cells exposed for 24 h to peptide-pulsed autologousEBV-transformed B cells, in IL-2-free medium at various peptideconcentrations, were screened for secretion of IFN-γ using an ELISA kit(R & D Systems, Minneapolis, Minn.). The results were expressed inpg/ml. A CBA system (BD PharMingen, San Diego, Calif.) was also used todetermine the secretion of multiple cytokines by specific T cells. TheCBA system uses the fluorescence detection by flow cytometry to measuresoluble analytes in a particles-based immunoassay. The BD human Th1/Th2cytokine CBA Kit was used to measure IL-2, IL-4, IL-5, IL-10, TNF-αprotein levels in a single sample. The cytokine capture beads were mixedwith PE-conjugated detection antibodies and then incubated withrecombinant cytokine standards or test samples to form sandwichcomplexes. The sample results were generated in graphic and tabularformat, using BD PharMingen CBA analysis software. The results wereexpressed in pg/ml.

Statistical Analysis.

Statistical analysis of differences between means was done using atwo-tailed paired t test (Stat View statistical software, AbacusConcepts, Berkeley, Calif.).

Toxicity Grading—the NCI Common Toxicity Scale.

-   -   If Grade I-II toxicities occur, the subject may continue with        the infusion schedule.    -   If Grade III toxicity occurs, the “drug” is held until the        toxicity decreases to Grade I or II, then the infusion is        restarted. If Grade III or IV toxicity occurs after the restart,        the “drug” infusions are stopped.    -   If Grade IV toxicity occurs, the subject is scored as having        Grade IV toxicity and the next infusion is reduced to the        previous dose. If the previous dose causes Grade IV toxicity,        then the “drug” is stopped.    -   If Grade IV toxicity occurs in 1 of 3 subjects at a specific        dose level, an additional 3 subjects are entered at that        cell-dose level for a total of 6 subjects at that dose level. If        2 of 6 subjects at a cell-dose level develop Grade IV toxicity,        this dose is defined as the MTD. The next 3 subjects are given        66% (two-thirds) of the previous cell-dose level. For the        purposes of evaluation for dose-escalation, each subject at the        same dose level must have received at least 4 of 6 infusions.

Example 2 Novel MUC-1 Binding Motifs

The primary amino acid sequence of human MUC-1 was analyzed forconsensus motifs for novel HLA-A2 binding peptides. Twelve 10-merpeptides were identified, consequently synthesized, and studied forbinding to the HLA-A2 molecule in a T2 cell binding assay. The aminoacid sequences and positions of these 10-mer peptides are shown inTable 1. The CEA CAP 1-6D peptide and a NCA peptide were used as apositive and negative control, respectively. The predicted binding ofthe 12 peptides are also given in Table 1. Three of these peptides(P-92, P-94 and P-1108) were shown to have the highest level of bindingin the T2 assay.

TABLE 1 Binding of human MUC-1 peptides to HLA-A2 molecules Amino acidPredicted SEQ Position in binding to T2 ID Peptide MUC-1 SequenceHLA-A2* binding^(#) NO: P-92  92-101 ATWGQDVTSV POS 740 1 P-94  94-103WGQDVTSVPV NEG 591 8 P-1108 1108-1117 REGTINVHDV NEG 482 9 P-4  4-13GTQSPFFLLL NEG 467 10 P-1105 1105-1114 LAFREGTINV NEG 461 11 P-11041004-1013 TLASHSTKTD NEG 442 12 P-1069 1069-1078 LQRDISEMFL NEG 433 13P-1162 1162-1171 ALLVLVCVLV POS 431 3 P-1135 1135-1144 TISDVSVSDV POS422 4 P-1172 1173-1181 ALAIVYLIAL POS 372 5 P-1169 1169-1178 VLVALAIVYLPOS 369 6 P-1177 1177-1186 LIALAVCQC POS 338 7 CAP1-6D NA YLSGADLNL POS975 38 NCA NA YRPGENLNL NEG 365 39 *Predicted binding on the basis ofreported motif (37); POS = positive; NEG = negative. ^(#)Results areexpressed in relative fluorescence. CAP1-6D is an HLA-A2 bindingcarcinoembryonic antigen peptide that was used as a positive control.NCA peptide was used as a negative control. NA = not applicable.

Example 3 Establishment of MUC-1 Specific T Cell Lines

Studies were then conducted to determine if MUC-1-specific T-cell linescould be established from PBMCs of an apparently healthy donor. Toaccomplish this, autologous DCs infected with rF-MUC-1/TRICOM were usedas APC. rF-MUC-1/TRICOM is a replication-defective avipox vectorcontaining the transgenes for MUC-1 and for a triad of humancostimulatory molecules (B7-1, ICAM-1 and LFA-3, designated TRICOM).rF-MUC-1/TRICOM was shown to efficiently infect human DCs andhyperexpress each of the costimulatory molecules, as well as MUC-1, onthe DC surface (Table 2). Approximately 96% of the cells were CD11c andMHC-class II positive.

TABLE 2 Phenotypic analysis of DCs infected with rF-MUC-1/TRICOMDendritic cells infected with CD80 CD54 CD58 Class I MUC-1 Uninfected4.8 (13.6) 59.5 (62.3) 68.1 (18.2) 99.7 (271.8) 5.0 (95.7) FP/WT 7.4(13.3) 79.3 (83.4) 74.0 (19.5) 99.5 (176.3) 3.1 (50.7) rF-MUC- 30.9(35.7)   84.5 (133.9) 79.8 (27.4) 99.9 (189.3) 31.6 (213.1) 1/TRICOMFlow cytometric analysis of surface marker expression on DCs. DCs usedwere cultured in AIM-V medium containing 100 ng/ml of rhGM-CSF and 20ng/ml of rhIL-4 for 7 days. DCs used for infection with FP/WT orrF-MUC-1/TRICOM were cultured as described in “Materials and Methods.”Results indicate the percentage of positive cells; numbers inparentheses represent MFI.

The specificity of the MUC-1-specific T cells generated (designatedT-1191-MUC-1) was analyzed after IVS cycle 3 (see Materials and Methods)for their ability to release IFN-γ after stimulation with autologous Bcells pulsed with each of the MUC-1 peptides listed in Table 1. Theresults shown in Table 3 demonstrate that when the T-1191-MUC-1 cellswere stimulated with autologous B cells pulsed with peptides P-92,P-1135, P-94, P-1004, P-1069 and P-4, the T cells produced IFN-γ, whilethe use of autologous B cells pulsed with the other peptides did notresult in IFN-γ production. The results shown in Tables 1 and 3demonstrate that the P-92 peptide had the highest level of T2 binding,as well as the ability to activate T-1191-MUC-1 cells to produce thehighest levels of IFN-γ; this peptide was thus chosen for further study.

TABLE 3 Production of IFN-γ by T-1191-MUC-1 cells stimulated withautologous B cells pulsed with MUC-1 peptides Production of IFN-γ(pg/ml) Peptide With peptide No peptide P-92 380.8 <52.3 P-1135 347.2<52.3 P-94 323.6 <52.3 P-1004 305.6 <52.3 P-1069 288.0 <52.3 P-4 260.0<52.3 P-1105 <52.3 <52.3 P-1108 <52.3 <52.3 P-1162 <52.3 <52.3 P-1169<52.3 <52.3 P-1172 <52.3 <52.3 P-1177 <52.3 <52.3 T-1191-MUC-1 cellswere used as effectors at IVS-3. T cells were stimulated with irradiatedautologous EBV-transformed B cells pulsed with different MUC-1 peptidesat a concentration of 25 μg/ml, and an effector-to APC ratio of 1:3.Twenty-four-hour culture supernatants were collected and screened forthe secretion of IFN-γ.

Example 4 Peptide Analysis

Analysis of the primary and secondary HLA-A2 anchor amino acid residuesat positions 2 and 10 of the P-92 peptide revealed that modification ofamino acids at these positions could potentially enhance the bindingability of the peptide to the HLA-A2 molecule. Thus, six differentanalogues of P-92 were synthesized, as shown in Table 4, and tested fortheir binding ability to T2 cells along with the native P-92 peptide.The CEA CAP-7 (HLA-A3 binding peptide), which has previously been shownnot to bind to HLA-A2, was used as a negative control. As shown in Table4, four of the six analog peptides bound to HLA-A2 at higher levels thanthe P-92 peptide. Analogues P-93L and P-93I bound HLA-A2 with greatestefficiency.

TABLE 4 MUC-1 peptide analogues Amino acid SEQ ID sequenceInitial designation T2 binding* NO: ATWGQDVTSV P-92 (native) 510 1 A

WGQDVTSV I-93 823 14 A

WGQDVTSV L-93 821 19 A

WGQDVTS

L-93/L-101 736 15 A

WGQDVTSV M-93 723 16 A

WGQDVTS

M-93/L-101 325 17 A

WGQDVTS

I-93/L-101 280 18 Amino acid sequences of the parental P-92 peptide(amino acid positions 92-101 of MUC-1) and analogue peptides. Aminoacids are shown by the single-letter code. Substitution amino acids areindicated in bold and italic. *Results are expressed in relativefluorescence values. HLA-A3 peptide (T2 binding = 200) was used as anegative control and CAP1-6D (T2 binding = 875) was used as a positivecontrol.

Experiments were then conducted to compare the ability of the P-93L andP-93I peptides to bind HLA-A2 at various peptide concentrations. As seenin FIG. 1, the P-93L and P-93I peptides bound to HLA-A2 at higher levelsthan P-92 at all peptide concentrations. The levels of binding weresimilar for P-93L and P-931 at the various peptide concentrations. Thesedata thus indicated that both P-93L and P-93I with modification in theprimary anchor position 2 (position 93 of the MUC-1 molecule) werepotential agonists of peptide P-92.

Example 5 Stability of Peptide-MHC Complex

The stability of the peptide-MHC complex for peptides P-92 (native),P-93L and P-93I, was examined. Peptides were incubated with T2 cellsovernight, the unbound peptides were washed off, and then incubated withbrefeldin-A to block delivery of new class I molecules to the cellsurface; at various time points cells were analyzed for the presence ofpeptide-HLA-A2 complexes. As shown in FIG. 2, P-93L-HLA-A2 andP-93I-HLA-A2 complexes were more stable than P-92-HLA-A2 complexes overthe 8-hour observation period, with P-93L-HLA-A2 complexes slightly morestable than the P-931-HLA-A2 complexes over the same period of time.Thus, both the binding of peptides to MHC and the stability of thepeptide-MHC complex were shown to be greater for the P-93L and P-93Ipeptides than the native P-92 peptide.

The ability of the P-93L and P-93I agonist peptides at various peptideconcentrations, to activate the T-1191-MUC-1 cells, was compared. Asseen in FIG. 3, at each concentration of peptide, pulsing of APC withthe P-93L peptide led to the greatest level of IFN-γ production byT-1191-MUC-1 cells as compared with the P-93I peptide or the native P-92peptide. The P-93L agonist peptide was thus chosen for further study.The cytokine profile of T-1191-MUC-1 cells stimulated with APCs pulsedwith either the P-92 or the P-93L peptide was then analyzed. A CBA assaywas used for the analysis. Table 5 shows the levels of each of the sixcytokines produced by T-1191-MUC-1 cell line stimulated with APCs pulsedwith no peptide, P-92 peptide and P-93L peptide. These resultsdemonstrated greater production of type 1 cytokine IL-2 and IFN-g by Tcells stimulated with the P-93L peptide than with the P-92 peptide. Lowor undetectable levels of type 2 cytokines IL-4 and IL-10 were seen witheither peptide. No TNF-α could be detected in the supernatants at the24-h time point.

TABLE 5 CBA assay for the production of cytokines by MUC-1 peptide-stimulated T cells Cytokines Peptide IL-2 IL-4 IL-5 IL-10 TNF-α IFN-γNone <20 <20 <20 <20 <20 <20 P-92 58.8 <20 <20 <20 <20 266 P-93L 366.9<20 140.9 <20 <20 650 The production of IL-2, IL-4, IL-5, IL-10, TNF-α,and IFN-γ was analyzed. Standards at concentrations of each cytokine at0 pg/ml, 312 pg/ml and 5000 pg/ml were used to determine theconcentrations of these six cytokines in the samples. T-1191 MUC-1 cellsat IVS-3 were used as effectors. T cells were stimulated with irradiatedautologous EBV-transformed B cells pulsed without peptide or with P-92or P-93L peptides at a concentration of 25 μg/ml, and an effector-to-APCratio of 1:3. Twenty-four-hour culture supernatants were collected andscreened for the secretion of cytokines. Results are expressed inpg/ml/10⁶ cell/ml.

To further compare the biologic activity of the native P-92 peptide andthe agonist P-93L peptide, two additional T-cell lines were established.This was accomplished using as APC autologous DCs infected withrF-MUC-1/TRICOM and autologous PBMCs as effectors from an apparentlyhealthy donor. After three IVS, the T-cell lines were stimulated withautologous B cells pulsed with either the P-92 or the P-93L peptide.These two cell lines were designated T-1191-P-92 and T-1191-P-93L,respectively. The two cell lines were shown to be >98% CD8 positive, 99%CD49d positive, <2% CD56 positive and >75% CD45RO positive cells. Thetwo T-cell lines were then analyzed for their ability to lysepeptide-pulsed targets. T-1191-P-93L was shown to lyse C1R-A2 cellspulsed with P-93L peptide to a greater extent than cells pulsed with theP-92 peptide (FIG. 4, squares). T-1191-P-92 also lysed target cellspulsed with the P-93L peptide to a greater extent than those pulsed withthe P-92 peptide (FIG. 4, triangles). The data in FIG. 4 also show thatwhen target cells are pulsed with the native peptide, the T-cell lineestablished with the agonist P-93L peptide lyses target cells at greaterlevels than the T-cell line established with the native peptide. Thiswas seen at two different E:T ratios. T-1191-P-93L cells and T-1191-P-92cells did not lyse C1R-A2 cells when pulsed with control CEA CAP 1-6Dpeptide (FIG. 4, circles).

Example 6 Target Cell Recognition

Studies were the conducted to determine whether these two T-cell linescould lyse the MUC-1 positive and HLA-A2 positive breast carcinoma cellline MCF-7. The MUC-1 negative HLA-A2 positive SK-Mel-24 melanoma cellline was used as a negative control. As shown in Table 5, MCF-7 cellswere lysed by both the T-1191-P-92 and T-1191-P-93L cells. No lysis wasobserved against the SK-Mel-24 cells. T-1191-P-93L cells were shown tolyse MCF-7 cells to a greater degree as compared with the T-1191-P-92cell line. The addition of unlabeled C1R-A2 cells pulsed with thecorresponding MUC-1 peptide, but not the CEA CAP1-6D control peptide,decreased the cytotoxic activity of both T-cell lines, demonstrating theMUC-1 specificity of the lysis (Table 5). The cytotoxic activity ofthese T-cell lines against MCF-7 cells was also shown to beHLA-A2-restricted, as demonstrated by the inhibition of lysis with theaddition of anti-HLA-A2 antibody, but not with the control antibodyUPC-10 (Table 6).

TABLE 6 Ability of MUC-1-specific T-cell lines T-1191-P-92 andT-1191-P-93L to lyse a MUC-1-expressing tumor cell line (MCF-7) TargetT-1191-P-92 T-1191-P-93L MCF-7 16.5 (2.7)* 24.5 (4.5)* MCF-7 + C1R-A215.6 (3.2)* 21.6 (2.9)* MCF-7 + C1R-A2 + P-92  4.2 (2.2)  6.1 (1.9)MCF-7 + C1R-A2 + P-93L  3.0 (1.5)  3.5 (1.2) MCF-7 + C1R-A2 + CAP1-6D17.1 (3.8)* 20.8 (3.9)* SK-Mel-24  0.5 (1.1)  1.4 (0.8) Results areexpressed in % of specific lysis at E:T = 25:1. The numbers inparentheses are the standard deviation. MCF-7 (human breast carcinomacell line) cells are MUC-1 positive and HLA-A2 positive. SK-Mel-24(melanoma) cells are MUC-1 negative and HLA-A2 positive. T-1191-P-92cells and T-1191-P- 93L cells were used at IVS-5. The T-1191-P-92 cellline was passaged on the native P-92 peptide, while the T-1191-P-93 cellline was passaged on the agonist P-93L peptide, from IVS 3 to IVS 5.MCF-7 cells were labeled with ¹¹¹In. Labeled MCF-7 cells and unlabeledC1R-A2 cells were used at a ratio of 1:10. C1R-A2 cells were incubatedwith or without P-92 peptide (25 μm/ml), P-93L peptide (25 μg/ml) orCAP1-6D control peptide (25 μg/ml). *Statistically significant lysis (p< 0.01, two-tailed t test) when comparing lysis of MCF-7 cells versusSK-Mel-24 cells. There is also a statistical significance (p < 0.01,two-tailed t test) when comparing lysis of MCF-7 + C1R-A2 versus MCF-7 +C1R-A2 + P-92 peptide or MCF-7 + C1R-A2 versus MCF-7 + C1R-A2 + P-93L.

The cytotoxic activity of these T-cell lines against MCF-7 cells wasalso shown to be HLA-A2-restricted, as demonstrated by the inhibition oflysis with the addition of anti-HLA-A2 antibody, but not with thecontrol antibody UPC-10 (Table 7).

The T-1991-P-92 and T-1191-P-93L T-cell lines were derived from anapparently healthy subject. Studies were then conducted to determine ifadditional T-cell lines could be established from two subjects withpancreatic cancer (subjects 23 and 18). Four MUC-1-specific T-cell lineswere generated and were designated T-23-P-92, T-23-P-93L, T-18-P-92 andT-18-P-93L. The T-cell lines T-18-P-92 and T-18-P-93L were generatedfrom subject 18 by stimulation of PBMCs with autologous DCs pulsed withthe P-92 and P-93L peptides, respectively. T-cell lines T-23-P-92 andT-23-P-93L were generated from subject 23 by stimulation of PBMCs byautologous DCs pulsed with the P-92 and P-93L peptides, respectively. Asseen in Table 8, all four T-cell lines from both pancreatic cancersubjects could be stimulated to produce IFN-γ when stimulated with DCspulsed with either the P-92 or the P-93L peptide. No IFN-γ productionwas observed, however, when these T cells were stimulated in a similarmanner with the CEA peptide CAP 1-6D. For all four T-cell lines, greaterlevels of IFN-γ production were seen when the P-93L agonist peptide wasused as compared with the P-92 native peptide. It also should be notedthat the T-cell lines derived using the agonist peptide always showedhigher levels of stimulation than the T-cell lines derived from thenative peptide, when a given peptide was used for stimulation.

TABLE 8 Production of IFN-γ, by T-cell lines generated from twopancreatic cancer subjects, stimulated with P-92, and agonist P-93Lpeptide Peptide T-cell line P-92 P-93L CAP1-6D T-23-P-92 299.8 644.5 <26T-23-P-93L 400.5 973.0 <26 T-18-P-92 168.0 366.6 <26 T-18-P-93L 378.2524.1 <26 ^(a)Results are expressed as pg IFN-γ produced. Cells fromfour MUC-1-specific T-cell lines established from two pancreatic cancersubjects (subjects 23 and 18) were used as effector cells at IVS-4.These T-cell lines were established by stimulation with P-92-pulsedautologous DCs (T-23-P-92 and P-18-P-92) or P-93L pulsed autologous DCs(T-23-P-93L and T-18-P-93L). For IFN-γ production, T-cell lines werestimulated with irradiated HLA-A-positive allogeneic DCs pulsed witheither P-92 or P-93L peptide at a concentration of 25 μg/ml and aneffector-to-APC ratio of 10:1. Twenty-four-hour culture supernatantswere collected and screened for the secretion of IFN-γ.

Example 7 Cytolysis of Targets by Cancer Subject Derived Cell Lines

Studies were then conducted to determine if the T-cell lines derivedfrom cancer subject 23 could lyse MUC-1 positive HLA-A2 positive cancercells. The SK-MeI-24 cell line (MUC-1 negative and HLA-A2 positive) wasused as a negative control for specificity. As can be seen in Table 9and 10, the T-23-P-93L line, T-23-P-92 line, T-18-P-93L line andT-18-P-92 line showed lysis of the MCF-7 carcinoma cells at twodifferent E:T ratios, but showed no lysis of the melanoma cell line. Inconcordance with the results shown above, the T-cell line (T-23-P-93L,T-18-P-93L) derived using the agonist peptide demonstrated greater lysisof the tumor cells than the T-cell line (T-23-P-92, T-18-P-92) derivedusing the native peptide. This was seen at two different E:T ratios.

TABLE 9 Ability of T-cell lines from a pancreatic cancer subject,generated with agonist peptide P-93L, to lyse cancer cells expressingnative MUC-1 E:T Ratios T-cell line Target 25:1 12.5:1 T-23-P-93L MCF-724.6 (1.0)*^(#) 17.9 (0.2)*^(#) T-23-P-93L SK-Mel-24  1.3 (1.1)  0.8(0.6) T-23-P-92 MCF-7 14.4 (0.6)* 10.1 (0.8)* T-23-P-92 SK-Mel-24  0.5(1.6)  0.4 (1.9) *Statistical significance (P < 0.01, two-tailed t test)when comparing lysis of MCF-7 cells versus SK-Mel-24 cells by T-23-P-93Land T-23P-92 cells. ^(#)Statistical significance (P < 0.01, two-tailed ttest) when comparing lysis of MCF-7 cells by T-23-P-93L and T-23-P-92cells. Results are expressed as % lysis.

TABLE 10 Ability of T cells from a pancreatic cancer subject generatedwith agonist peptide P-93L to lyse cancer cells expressing native MUC-1E:T Ratios T-cell line Target 25:1 12.5:1 T-18-P-93L MCF-7   25(0.8)*^(#) 14.6 (0.5)*^(#) T-18-P-93L SK-Mel-24  1.1 (0.9)  1.0 (0.8)T-18-P-92 MCF-7 12.1 (0.3)*  8.4 (0.4)* T-18-P-92 SK-Mel-24  0.9 (1.4) 1.0 (1.2) *Statistical significance (P < 0.01, two-tailed t test) whencomparing lysis of MCF-7 cells versus SK-Mel-24 cells by T-18-P-93L andT-18-P-92 cells. ^(#)Statistical significance (P < 0.01, two-tailed ttest) when comparing lysis of MCF-7 cells by T-18-P-93L and T-18-P-92cells.

Example 8 Materials and Methods

The two viral vectors analyzed are the replication competent recombinantvaccinia virus (rV-), and the avipox vector, fowlpox (rF-), which isreplication incompetent in mammalian cells. Each vector encodes thetransgenes for three human costimulatory molecules (B7-1, ICAM-1, LFA-3,designated TRICOM); both the CEA and MUC-1 transgenes also containagonist epitopes. The vectors are designated rV-CEA/MUC/TRICOM andrF-CEA/MUC/TRICOM.

Each of the vectors is shown to be capable of faithfully expressing allfive transgenes in human dendritic cells (DCs). DCs infected with eithervector are shown to activate both CEA-specific and MUC-1-specific T-celllines to the same level as DCs infected with CEA-TRICOM or MUC-1-TRICOMvectors. Thus, no evidence of antigenic competition between CEA andMUC-1 was observed. Human DCs infected with rV-CEA/MUC/TRICOM orrF-CEA/MUC/TRICOM are also shown to be capable of generating both MUC-1and CEA-specific T-cell lines; these T-cell lines are in turn shown tobe capable of lysing targets pulsed with MUC-1 or CEA peptides, as wellas human tumor cells endogenously expressing MUC-1 and/or CEA.

Cell Cultures

The human breast adenocarcinoma cell line MCF-7 (HLA-A2 positive, CEAnegative and MUC-1 positive), the colorectal carcinoma cell line SW1463(HLA-A2 positive, CEA positive and MUC-1 positive), and the melanomacell line SKMel-24 (HLA-A2 positive, CEA negative and MUC-1 negative)were purchased from American Type Culture Collection (Manassas, Va.).The cultures were free of mycoplasma and were maintained in completemedium [RPMI 1640 (Invitrogen Life Technologies, Inc., Carlsbad, Calif.)supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mlpenicillin, and 100 μg/ml streptomycin (Invitrogen Life Technologies)].The C1R cell line is a human plasma leukemia cell line that does notexpress endogenous HLA-A or B antigens. C1R-A2 cells are C1R cells thatexpress a transfected genomic clone of HLA-A2.1. These cells wereobtained from Dr. William E. Biddison (National Institute ofNeurological Disorders and Stroke, NIH, Bethesda, Md.). The 174CEM-T2cell line (T2) transport deletion mutant was provided by Dr. PeterCresswell (Yale University School of Medicine, New Haven, Conn.). C1R-A2cells and T2 cells were mycoplasma free and were maintained in RPMI 1640complete medium and Iscove's modified Dulbecco's complete medium(Invitrogen Life Technologies), respectively. The V8T cell line is aCD8+ CTL line directed against the CAP-1 epitope of CEA. The T-1191-P93Lcell line is a CD8+ MUC-1-specific CTL line generated from peripheralblood mononuclear cells (PBMCs) from a healthy donor that was in vitrostimulated using a MUC-1 peptide. Both V8T and T-1191-P93L cell lineswere cultured as described previously.

Peptides

HLA-A2 binding peptides used included: (a) the CEA agonist peptide CAP1-6D (YLSGADLNL) (SEQ ID NO: 38), designated CEA peptide, (b) the MUC-1agonist peptide P-93L (ALWGQDVTSV) (SEQ ID NO: 2), designated MUC-1peptide, (c) the prostate-specific antigen (PSA) peptide PSA-3(VISNDVCAQV) (SEQ ID NO: 40). All peptides were greater than 96% pureand manufactured by American Peptide Company, Inc. (Sunnyvale, Calif.).

Culture of DCs from PBMCs

HLA-A2 normal donor PBMCs were obtained from heparinized blood. PBMCswere separated using lymphocyte separation medium gradient (OrganonTeknika, Durham, N.C.), as described previously (51) (Boyum A. Aone-stage procedure for isolation of granulocytes and lymphocytes fromhuman blood. General sedimentation properties of white blood cells in 1g gravity field, Scand J Clin Lab Invest 1968; 97(Suppl):51-76). DCswere prepared using a modification of the procedure described bySallusto et al. Sallusto F, Lanzavecchia A., Efficient presentation ofsoluble antigen by cultured human dendritic cells is maintained bygranulocyte/macrophage colony stimulating factor plus interleukin-4 anddown-regulated by tumor necrosis factor alpha., J Exp Med 1994;179:1109-18. PBMCs (1.5×10⁸) were resuspended in AIM-V medium containing2 mM glutamine, 50 μg/ml streptomycin, and 10 μg/ml gentamycin(Invitrogen Life Technologies), and allowed to adhere to a T-150 flask(Corning Costar Corp., Cambridge, Mass.). After 2 hours at 37° C., thenon-adherent cells were removed with a gentle rinse. The adherent cellswere cultured for 6-7 days in AIM-V medium containing 100 ng/ml ofrecombinant human GM-CSF (rhGM-CSF) and 20 ng/ml of recombinant humanIL-4 (rhIL-4). The culture medium was replenished every 3 days.

Recombinant Virus and Infection of DCs with rV-CEA/MUC/TRICOM andrF-CEA/MUC/TRICOM

Both rV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM encode the human CEA genecontaining the 6D modification, the human MUC-1 gene containing the 93Lmodification and the genes for the human costimulatory molecules B7-1,ICAM-1, and LFA-3 (FIG. 5) (Zaremba S, Barzaga E, Zhu M et al.Identification of an enhancer agonist cytotoxic T lymphocyte peptidefrom human carcinoembryonic antigen. Cancer Res 1997; 57:4570-7, andTsang K Y, Palena C, Gulley J, Arlen P, Schlom J. A human cytotoxicT-lymphocyte epitope and its agonist epitope from the non-variablenumber of tandem repeat sequence of MUC-1. Clin Cancer Res 2004;10:2139-49) Recombinant vectors were generated by homologousrecombination as described previously (Hodge J W, McLaughlin J P, KantorJ A, Schlom J. Diversified prime and boost protocols using recombinantvaccinia virus and recombinant nonreplicating avian pox virus to enhanceT-cell immunity and antitumor responses. Vaccine 1997; 16:759-68). DCs(1×106) were incubated in 1 ml of Opti-MEM medium (Invitrogen LifeTechnologies) at 37° C. with rF-CEA/MUC/TRICOM, rV-CEA/MUC/TRICOM,control avipox virus vector (FP-WT) or control vaccinia vector (V-WT).Titration experiments demonstrated that infection of DCs for 2 hourswith 4×10′ plaque-forming units (pfu)/ml of rF-CEA/MUC/TRICOM, equal toa multiplicity of infection (MOI) of 40 pfu/cell was able toconsistently induce transgene expression in approximately 60% of theinfected DCs. Similar titration experiments demonstrated that infectionof DCs for 1 hour with 0.5×10⁷ pfu/ml of rV-CEA/MUC/TRICOM, equal to anMOI of 5 pfu/cell, was able to consistently induce transgene expressionin approximately 35% of the infected DCs. DCs from different donors wereused for the infections with rF-CEA/MUC/TRICOM and rV-CEA/MUC/TRICOM,with the efficiency of infection ranging from 50%-65% forrF-CEA/MUC/TRICOM and 30%-59% for rV-CEA/MUC/TRICOM. The infected DCswere suspended in 10 ml of fresh, warm RPMI-1640 complete mediumcontaining 100 ng/ml of rhGM-CSF and 20 ng/ml of rhIL-4, cultured for 24hours, and subsequently used as APC.

Flow Cytometric Analysis

Dual-color flow cytometric analysis was performed on Tcell lines byusing the following antibody combinations: anti-CD56-FITC/anti-CD8-PE,anti-CD8-FITC/anti-CD45RA-FITC, and anti-CD8-FITC/anti-CD27-PE.Antibodies were all purchased from BD Biosciences (San Jose, Calif.).Staining was conducted simultaneously for 1 hour at 4° C., cells werethen washed three times with cold Ca2⁺ and Mg2⁺ free phosphate-bufferedsaline (PBS), resuspended in the same buffer, and 11 immediatelyanalyzed using a FACScan and the CELLQuest program (BD Biosciences).Data were gathered from 10,000 live cells, stored, and used to generateresults. The procedure for analysis of DCs was similar to the onedescribed above. The following antibody combinations were used:anti-MHC-class II-FITC/anti-CD80-PE, anti-CD58-FITC/anti-CD54-PE,anti-MHC class I-FITC/anti-MHC class II-PE, andanti-IgG1-FITC/anti-IgG2a-PE (isotype controls). Antibodies to MHC-classI and II were purchased from Serotec (Oxford, UK); other antibodies werepurchased from BD Biosciences. The anti-CEA monoclonal antibody COL-1and anti-MUC-1 antibodies (DF3 and DF3-P) were also used (Muraro R,Wunderlich D, Thor A, et al. Definition by monoclonal antibodies of arepertoire of epitopes on carcinoembryonic antigen differentiallyexpressed in human colon carcinoma versus normal adult tissues. CancerRes 1985; 45:5769-80). MOPC-104E (IgM) (Cappel/Organon Teknika Corp.,West Chester, Pa.) was used as negative control.

After staining, cells were washed three times and subsequently incubatedwith a 1:100 dilution of FITC-labeled goat anti-mouse immunoglobulin(IgG) (Kirkegaard and Perry Laboratories, Gaithersburg, Md.). Analysiswas conducted as described above. Results were expressed in percentageof positive cells and mean fluorescence intensity (MFI). The MFI valuewas collected in log scale, and was used to express the levels offluorescence determined by measuring the average for all the cells inthe fluorescence dot plot.

Immune Blot Analysis

Uninfected DCs, DCs infected with 40 MOI of rF-CEA/MUC/TRICOM,rF-CEA(6D)-TRICOM or rF-MUC-1-TRICOM vectors, and DCs infected with 5MOI of the rV-CEA/MUC/TRICOM vector were lysed by using the MPERMammalian Protein Extraction Reagent (Pierce, Rockford, Ill.). Proteinconcentration of the lysates was determined by using a MicroBCA ProteinAssay Kit (Pierce), and 20 μg-fractions of protein per sample wereblotted onto a PVDF membrane 12 using a Bio-Dot Microfiltrationapparatus (BioRad Laboratories, Hercules, Calif.), following themanufacturer's instructions. After blotting, the membranes were blockedfor 1 hour at room temperature with PBS containing 5% BSA (BiosourceInternational, Camarillo, Calif.). Membranes were then washed threetimes with PBS containing 0.25% Tween-20, and incubated for 2 hours atroom temperature with a solution at 1 μg/ml of COL-1, DF-3 or DF3-Pantibodies. Membranes were then washed three times as above, andincubated with a 1:3000 dilution of an anti-mouse IgG conjugated to HRP(Kirkegaard & Perry Laboratories) for 1 hour at room temperature. Forimmunodetection of the CEA and MUC-1 proteins, the SuperSignal West PicoChemiluminescent Substrate was used (Pierce).

Generation of T-Cell Lines

Modification of the protocol described by Tsang et al. was used togenerate CEA and/or MUC-1-specific CTL (Tsang K Y, Zaremba S, Nieroda CA, et al. Generation of human cytotoxic T cells specific for humancarcinoembryonic antigen epitopes from patients immunized withrecombinant vaccinia-CEA vaccine. J Natl Cancer Inst 1995; 87:982-90).To generate T-cell lines T-rV and T-rF, autologous DCs infected withrV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM, respectively, were used as APC.Autologous non-adherent cells were added to APC at an effector-to-APCratio of 10:1; cultures were incubated for 3 days at 37° C., in ahumidified atmosphere containing 5% CO². The cultures were thensupplemented with recombinant human IL-2 at a concentration of 20units/ml for 7 days; the IL-2 containing medium was replenished every 3days. The 3-day incubation with peptide and 7-day IL-2 supplementconstituted one in vitro stimulation (IVS) cycle. T-rV and T-rF wererestimulated with rV-CEA/MUC/TRICOM- or -CEA/MUC/TRICOM-infectedautologous DCs, respectively, as described above, on day 11 to begin thenext IVS cycle. rV-CEA/MUC/TRICOM- and rF-CEA/MUC/TRICOM-infectedautologous DCs were used as APC for three IVS cycles. For the generationof T-rF(CEA) and T-rF(MUC) cell lines, T cells were stimulated withautologous DCs infected with rF-CEA/MUC/TRICOM for one IVS, and thenrestimulated with uninfected autologous DCs pulsed with CAP1-6D or P-93Lpeptide, respectively, for two more IVS. After the third IVS cycle,irradiated (23,000 rads) autologous EBV-transformed B cells were used asAPC. The EBV-transformed B cells were pulsed with 25 ∝cg/ml of peptide,and used for restimulation at an effector-to-APC ratio of 1:3.

Cultures were then incubated for 3 days at 37° C. in a humidifiedatmosphere containing 5% CO₂. After removal of the peptide containingmedium, the cultures were supplemented with recombinant human IL-2 at aconcentration of 20 units/ml for 7 days. T-cell lines from patients 55,49 and 41 were generated by stimulation of PBMCs with autologous DCsinfected with rF-CEA/MUC/TRICOM, using the same stimulation protocoldescribed above. Patient 55 initially underwent a Whipple procedure forlocalized pancreatic cancer followed by adjuvant radiation therapy tothe pancreatic bed. The patient had local recurrence and receivedchemotherapy with 5FU/Leucovorin followed by an experimental vaccinestudy using both vaccinia-CEA and ALVAC-CEA prior to enrolling on thisclinical trial. Patient 41 was diagnosed with colorectal carcinoma withliver metastasis. Prior to enrolling on study, this patient progressedon three different chemotherapy regimens, including5FU/Leucovorin/CPT-11, 5FU/Leucovorin/Oxalliplatin, and XELODA™(Capecitabine). Patient 49 had colorectal cancer with both liver andlung metastasis. The patient progressed following four cycles ofchemotherapy with CPT-11/5FU/Leucovorin prior to enrolling on study.

Cytotoxic Assay

Target cells (C1R-A2 or tumor cells) were labeled with 50 μCi of¹¹¹Indium-labeled oxyquinoline (Medi-Physics Inc., Arlington, Ill.) for15 minutes at room temperature. Target cells (0.3×10⁴) in 100 n1 ofRPMI-1640 complete medium were added to each of 96 wells inflat-bottomed assay plates (Corning Costar Corp.). Labeled C1R-A2 targetcells were incubated with peptides at the concentration indicated for 60minutes at 37° C. in 5% CO₂ before adding effector cells. No peptide wasused when carcinoma cell lines were used as targets. Effector cells weresuspended in 100 μl of RPMI-1640 complete medium supplemented with 10%pooled human AB serum and added to the target cells. The plates werethen incubated at 37° C. in 5% CO₂ for 6 or 16 hours. Supernatant washarvested for gamma counting with the use of harvester frames (Skatron,Inc., Sterling, Va.). Determinations were carried out in triplicate, andstandard deviations were calculated. Specific lysis was calculated withthe use of the following formula (all values in cpm):

% lysis=(Observed release−Spontaneous release/Total release−Spontaneousrelease)×100.

Spontaneous release was determined from wells to which 100 μl ofRPMI-1640 complete medium was added. Total releasable radioactivity wasobtained after treatment of targets with 2.5% Triton x-100.

Detection of Cytokines

Supernatants of T cells exposed for 24 hours to peptide-pulsedautologous EBV-transformed B cells in IL-2-free medium, at variouspeptide concentrations, were screened for secretion of IFN-α using anELISA kit (Biosource International). The results were expressed inpg/ml.

Statistical Analysis

Statistical analysis of differences between means was done using atwo-tailed paired t test (Stat View statistical software, AbacusConcepts, Berkeley, Calif.).

Studies were first undertaken to determine if infection of human DCswith rV-CEA/MUC/TRICOM would result in the expression of each of thefive transgenes. Initial studies used an MOI of 5 and 10 forrV-CEA/MUC/TRICOM; both MOI gave comparable results and thus an MOI of 5was used in subsequent experiments. As seen in FIG. 6, uninfected humanDCs do not express CEA (as detected by monoclonal antibody COL-1);expression of CD80, CD54, and CD58, MHC Class I and MHC Class II by DCsis similar to that previously reported in several studies (Tsang K Y,Zhu M Z, Even J., et al., The infection of human dendritic cells withrecombinant avipox vectors expressing a costimulatory molecule transgene(CD80) to enhance the activation of antigen-specific cytotoxic T cells.Cancer Res 2001; 61:7568-76; and Zhu M Z, Terasawa H, Gulley J, et al.Enhanced activation of human T cells via avipox vector-mediatedhyperexpression of a triad of costimulatory molecules in human dendriticcells. Cancer Res 2001; 61:3725-34). Infection with V-WT had little, ifany, effect on any of these eight surface markers (FIG. 6). Infectionwith rV-CEA/MUC/TRICOM, however, is shown to substantially increase thelevel of expression of CEA, MUC-1, CD80, CD54 and CD58, and did notmeasurably affect the level of expression of MHC Class I and Class II.Consistent with results previously published, infection of DCs from thesame donor with rV-CEA(6D)-TRICOM enhanced the level of CEA, CD80, CD54,and CD58 to levels similar to those seen with rV-CEA/MUC/TRICOM, but didnot alter the expression of MUC-1 or MHC Class I or Class II. Inaddition, infection of DCs with rV-MUC-1 admixed with rV-TRICOM(rV-MUC-1-TRICOM construct was not available) showed levels of enhancedexpression of MUC-1 and the three costimulatory molecules similar tothose seen with rV-CEA/MUC/TRICOM, but had no effect on the level ofexpression of MHC Class I and Class II, and on the lack of expression ofCEA (data not shown). Low levels of MUC-1 were detected in theuninfected and control vector-infected DCs. This is in agreement withthat reported by Wykes et al., which showed that MUC-1 is expressed onhuman DCs and monocytederived DCs when cultured in vitro (Wykes M,MacDonald K P, Tran M, et al. MUC1 epithelial mucin (CD227) is expressedby activated dendritic cells. J Leukoc Biol 2002; 72:692-701.)

Parallel studies were undertaken to determine if infection of human DCswith rF-CEA/MUC/TRICOM would result in the expression of each of thefive transgenes. Initial studies used an MOI of 20 and 40 forrF-CEA/MUC/TRICOM; an MOI of 40 showed greater expression of transgenesand was thus used in subsequent experiments. As seen in FIG. 7,infection with FP-WT had little, if any, effect on any of the eightsurface markers analyzed. Infection with rF-CEA/MUC/TRICOM, however, wasshown to substantially increase the level of expression of CEA, MUC-1,CD80, CD54 and CD58, but did not affect the level of expression of MHCClass I and Class II. Infection of DCs from the same donor withrF-CEA(6D)-TRICOM enhanced the level of CEA, CD80, CD54, and CD58 tosimilar levels as seen with rF-CEA/MUC/TRICOM, but did not alter theexpression of MUC-1 or MHC Class I or Class II. In addition, infectionof DCs with rF-MUC-1-TRICOM showed similar levels of enhanced expressionof MUC-1 and the three costimulatory molecules as seen withrF-CEA/MUC/TRICOM, but had no effect on the level of expression of MHCClass I and Class II and the lack of expression of CEA (data not shown).The expression of CEA and MUC-1 on DCs infected with rF-CEA/MUC/TRICOM,rF-CEA(6D)-TRICOM, rF-MUC-1-TRICOM vector or uninfected DCs was analyzedby immune blot analysis. As shown in FIG. 8, CEA was detected in DCsinfected with rF-CEA(6D)-TRICOM, rF-CEA/MUC/TRICOM, andrV-CEA/MUC/TRICOM, but not in uninfected DCs or rF-MUC-1-TRICOM-infectedDCs. As described above, DCs express a low level of MUC-1, but a greatincrease in MUC-1 expression was clearly observed in DCs infected withrF-MUC-1-TRICOM, rV-CEA/MUC/TRICOM, and rF-CEA/MUC/TRICOM, but not inDCs infected with rF-CEA(6D)-TRICOM (FIG. 8).

We have demonstrated the ability of DCs pulsed with the CEA agonistpeptide CAP-1 (6D) and the MUC-1 agonist peptide P-93L to activate humanT cells. Studies were undertaken to determine if infection of human DCswith the rF-CEA/MUC/TRICOM vector could stimulate IFN-γ production byCEA-specific and MUC-1-specific T cells. These results were alsocompared with the ability of human DCs infected with rF-CEA(6D)-TRICOMor rF-MUC-1-TRICOM to activate these T cells. As seen in Table 11,uninfected DCs or DCs infected with FP-WT did not result in any IFN-γproduction by the CEA-specific T-cell line (V8T), or the MUC-1-specificT cell line (T-1191-P93L). DCs pulsed with the CEA peptide induced IFN-γproduction only by the CEA-specific T-cell line, while DCs pulsed withthe MUC-1 peptide induced IFN-γ production only by the MUC-1-specificT-cell line. Similarly, DCs infected with rF-CEA(6D)-TRICOM inducedIFN-γ production only by the CEA-specific T-cell line, while DCsinfected with the rF-MUC-1-TRICOM induced IFN-γ production only by theMUC-1-specific T-cell line. Infection of DCs with rF-CEA/MUC/TRICOM,however, induced IFN-γ production in both the CEA-specific T-cell lineand the MUC-1-specific T-cell line, and at comparable levels to thoseseen when using the vectors containing only the single tumor-antigentransgene. These studies may demonstrate the lack of antigeniccompetition between CEA and MUC-1 in the rF-CEA/MUC/TRICOM vector in theability to activate T cells.

Studies were then undertaken to determine if infection of human DCs withthe rV-CEA/MUC/TRICOM vector could stimulate IFN-γ production 19 by theCEA-specific and MUC-1-specific T cells. These results were alsocompared with the ability of human DCs infected with rV-CEA(6D)-TRICOM,or rV-MUC-1 plus rV-TRICOM, to activate those T cells. As seen in Table12, uninfected DCs or DCs infected with V-WT did not result in any IFN-γproduction by the CEA-specific T-cell line or the MUC-1-specific T-cellline. DCs pulsed with the CEA peptide induced IFN-γ production only bythe CEA-specific T-cell line, while DCs pulsed with the MUC-1 peptideinduced IFN-γ production only by the MUC-1-specific T-cell line.Similarly, DCs infected with rV-CEA(6D)-TRICOM induced IFN-γ productiononly by the CEA-specific T-cell line, while DCs infected with the MUC-1vectors induced IFN-γ production only in the MUC-1-specific T-cell line.Infection of DCs with rV-CEA/MUC/TRICOM, however, induced IFN-γproduction in both the CEA-specific T-cell line and the MUC-1-specificT-cell line and at comparable levels to those seen when using thevectors containing only the single tumor-antigen transgene. Thesestudies may demonstrate the lack of antigenic competition between CEAand MUC-1 in the ability to activate T cells employing therF-CEA/MUC/TRICOM vector. Studies were then undertaken to determine ifhuman T cells could be established from PBMCs using autologous DCsinfected with rF-CEA/MUC/TRICOM and/or rV-CEA/MUC/TRICOM as APC.

After three IVS, as described in the Materials and Methods section,resultant T cells were analyzed for their ability to be activated by DCspulsed with peptides or infected with vector. As seen in Table 13,neither of the Tcell lines established using rV-CEA/MUC/TRICOM- orrF-CEA/MUC/TRICOM-infected DCs as APC could be activated to produceIFN-γ, when stimulated by uninfected 20 DCs, or DCs infected with FP-WT.These results are consistent with previous observations in the murinesystem that there is no cross-reactivity in terms of T-cell epitopesbetween vaccinia virus and fowlpox (Hodge J W, Poole D J, Aarts W M, etal. Modified vaccinia virus ankara recombinants are as potent asvaccinia recombinants in diversified prime and boost vaccine regimens toelicit therapeutic antitumor responses. Cancer Res 2003; 63:7942-9); itis also consistent with previous murine and human studies (unpublisheddata), which showed it is difficult to generate mammalian T cellsdirected against fowlpox epitopes. On the other hand, the T-cell linesgenerated with rV-CEA/MUC/TRICOM- or rF-CEA/MUC/TRICOM-infected APC wereboth activated to produce IFN-γ when using as APC DCs pulsed with eitherthe CEA or the MUC-1 peptides (Table 13). These results may indicatethat DCs infected with either vector will generate T cells from PBMCsthat are directed against both the CEA and MUC-1 antigens.

T cells generated by DCs infected with either rV-CEA/MUC/TRICOM orrF-CEA/MUC/TRICOM produced IFN-γ when exposed to DCs infected withrF-CEA/MUC/TRICOM. In additional experiments, T cells were generatedinitially employing rF-CEA/MUC/TRICOM-infected DCs as APC, and were thenpassaged with CEA peptidepulsed APC for two IVS. As seen from Table 13,these T cells lost their ability to be activated by DCs pulsed with theMUC-1 peptide, but retained their ability to be activated to produceIFN-γ by DCs pulsed with the CEA peptide. Conversely, when T cellsgenerated initially using rF-CEA/MUC/TRICOM-infected DCs were thenpassaged in the presence of DCs pulsed with MUC-1 peptide, they losttheir ability to be activated by DCs pulsed with CEA peptide, butretained their ability to be activated by MUC-1 peptide.

Studies were then conducted to determine if T-cell lines, establishedusing as APC DCs infected with either the rV-CEA/MUC/TRICOM orrF-CEA/MUC/TRICOM vector, could lyse human target cells. As seen inTable 14, both T-cell lines were unable to lyse CIR-A2 cells, but couldlyse C1R-A2 cells pulsed with either the CEA peptide or the MUC-1peptide. Neither T-cell line could lyse C1R-A2 cells pulsed with thecontrol PSA peptide. On the other hand, the T-cell line establishedusing DCs infected with rF-CEA/MUC/TRICOM, and then passaged using asAPC, CEA peptide-pulsed DCs, was able to lyse target cells pulsed withthe CEA peptide but not target cells pulsed with MUC-1 or PSA peptides.Conversely, the T-cell line established using DCs infected withrF-CEA/MUC/TRICOM, and then passaged using as APC, MUC-1 peptide-pulsedDCs, was able to lyse target cells pulsed with the MUC-1 peptide but nottarget cells pulsed with CEA or PSA peptides.

Studies were then conducted to determine if the T-cell lines generatedusing DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM vectorshad the ability to lyse human tumor cells expressing either native CEAor native MUC-1. Three HLA-A2+ target cell lines were evaluated: theMCF-7 human breast carcinoma line, which is positive for MUC-1 andnegative for CEA; the human colon carcinoma cell line SW1463, which ispositive for both CEA and MUC-1; and the SK-MeI-24 human melanoma line,which is negative for both MUC-1 and CEA expression. We were unable toidentify an HLA-A2+ cell line that was negative for MUC-1 and positivefor CEA. As seen in Table 14, the T-cell lines generated using DCsinfected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM both were able tolyse the breast and colon carcinoma lines, but were unable to lyse themelanoma line.

On the other hand, the T-cell line generated using DCs infected withrF-CEA/MUC/TRICOM and then restimulated with DCs pulsed with the CEApeptide for two IVS was able to lyse the CEA positive/MUC-1 positivecolon carcinoma line, but was unable to lyse the CEA negative/MUC-1positive breast cancer line and the CEA negative/MUC-1 negative melanomaline. The T-cell line generated using DCs infected withrF-CEA/MUC/TRICOM and then restimulated with DCs pulsed with the MUC-1peptide for two IVS was able to lyse both the colon and breast celllines, but not the melanoma line. Collectively, these data maydemonstrate that both recombinant vaccinia and avipox vectors can beconstructed to each faithfully express five human transgenes, and thatno antigenic competition is observed in the ability of these vectors toactivate human T cells directed against two human tumor-associatedantigens.

The T-rV, T-rF, T-rF(CEA) and T-rF(MUC) T-cell lines were generated froman apparently healthy individual. All four cell lines were shown tobe >97% CD8 positive, <2% CD56 positive, >75% CD45RA positive, and >81%CD27 positive. Studies were then conducted to determine whether specificT-cell lines could be derived from a patient with pancreatic cancer(patient 55). A T-cell line was generated usingrF-CEA/MUC/TRICOM-infected DCs as APC, and was designated T-55. Asdetermined by flow cytometric analysis, the T-55 cell line was 99.9% CD8positive, <2% CD56 positive, 73.6% CD45RA positive and 87% CD27positive. As seen in Table 15, this Tcell line was shown to produceIFN-γ when stimulated with autologous DCs infected withrF-CEA/MUC/TRICOM and DCs pulsed with either the CEA peptide or theMUC-1 peptide, but not the PSA-3 peptide.

Studies were then conducted to determine whether 23 this T-cell linecould lyse CEA and/or MUC-1 positive and HLA-A2 positive cancer celllines. The melanoma cell line SK-MeI-24 (MUC-1 negative, CEA negativeand HLA-A2 positive) was used as a negative control. As seen in Table16, the T-55 cell line showed lysis of the C1R-A2 cells pulsed with CEApeptide and MUC-1 peptide, but not PSA-3 peptide. In addition, the T-55cell line lysed MCF-7 cells and SW1463 cells at various E:T ratios, butshowed no lysis of the melanoma cell line. Two additional T-cell lineswere established from colon carcinoma patients. These T-cell lines weredesignated T-41 and T-49. The T-41 cell line was 98.8% CD8 positive, <1%CD56 positive, 33.6% CD45RA positive and 96.8% CD27 positive. The T-49cell line was 98.9% CD8 positive, <1% CD56 positive, 29.8% CD45RApositive and 95.3% CD27 positive. As seen in Table 15, both T-41 andT-49 cell lines were shown to produced IFN-γ when stimulated withautologous DCs infected with rF-CEA/MUC/TRICOM and DCs pulsed witheither the CEA peptide or the MUC-1 peptide, but not the PSA-3 peptide.As seen in Table 17, both T-41 and T-49 cell lines showed lysis of MCF-7and SW1463 at various E:T ratios, but showed no lysis of the SK-MeI-24cell line.

Two of the major concerns in the development and use of vaccines forcancer therapy are: (a) the poor immunogenicity of tumor-associatedantigens and (b) antigenic heterogeneity of tumors. The vectorsdescribed here were developed to address both these issues. Thesevectors are the first in which five complete transgenes are insertedinto an avipox vector and, to our knowledge, any replication incompetentvector. A parallel five transgene construct recombinant vaccinia virushas also been developed. For both vectors, each transgene was driven byits own promoter. Previous studies in both preclinical models andclinical trials have demonstrated that diversified prime and boostvaccine regimens using two different vaccines are superior to thecontinued use of one vaccine. It is for this reason that both therecombinant vaccinia and recombinant fowlpox vectors were developed.

As demonstrated in Tables 12-17, infection of DCs with therV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM vector resulted in the activationof T cells as efficiently as the use of DCs as APC that were infectedwith either CEA-TRICOM or MUC-1-TRICOM vectors. Moreover, T cellsgenerated using DCs infected with rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOMwere able to lyse target cells expressing either CEA or MUC-1.

TABLE 11 Production of IFN-γ by CEA-specific and MUC-1-specific T-celllines stimulated with rF-CEA/MUC/TRICOM IFN-γ (pg/ml) Treatment ofdendritic cells CEA-specific CTL MUC-1-specific CTL Uninfected <15 <15FP-WT <15 <15 Uninfected + CEA peptide 772 <15 Uninfected + MUC-1 <15458 peptide FP-WT + CEA peptide 689 <15 FP-WT + MUC-1 peptide <15 404rF-CEA(6D)-TRICOM 475 <15 rF-MUC-1-TRICOM <15 298 rF-CEA/MUC/TRICOM 455278 CEA-specific T cells (V8T) and MUC-1-specific T cells (T-1191-P93L)were stimulated with autologous uninfected DCs alone or pulsed witheither the CEA peptide (CAP1-6D) or the MUC-1 peptide (P-93L); DCsinfected with the control vector FP-WT alone or pulsed with either theCEA or MUC-1 peptides; DCs infected with rF-CEA/MUC/TRICOM, rF--CEA(6D)-TRICOM or rF-MUC-1/TRICOM. Peptides were used at a concentrationof 25 μg/ml. The effector-to-APC ratio was 10:1. Twenty-four hourculture supernatants were collected and screened for the production ofIFN-γ.

TABLE 12 Production of IFN-γ by CEA-specific and MUC-1-specific T-celllines stimulated with rV-CEA/MUC/TRICOM IFN-γ (pg/ml) Treatment ofdendritic cells CEA-specific CTL MUC-1-specific CTL Uninfected <15 <15V-WT <15 <15 Uninfected + CEA peptide 820 <15 Uninfected + MUC-1 <15 550peptide V-WT + CEA peptide 720 <15 V-WT + MUC-1 peptide <15 358rV-CEA(6D)-TRICOM 384 <15 rV-MUC-1 + rV-TRICOM <15 213 rV-CEA/MUC/TRICOM285 256 CEA-specific T cells (V8T) and MUC-1-specific T cells(T-1191-P93L) were stimulated with autologous uninfected DCs alone orpulsed with either the CEA peptide (CAP1-6D) or the MUC-1 peptide(P-93L); DCs infected with the control vector V-WT alone or pulsed witheither the CEA or MUC-1 peptides; DCs infected with rV-CEA/MUC/TRICOM,rV-CEA(6D)-TRICOM or rV-MUC-1 plus rV-TRICOM. Peptides were used at aconcentration of 25 μg/ml. The effector-to-APC ratio was 10:1.Twenty-four hour culture supernatants were collected and screened forthe production of IFN-γ.

TABLE 13 Production of IFN-γ by CEA- and MUC-1-specific T cellsestablished by using rV-CEA/MUC/TRICOM or rF-CEA/MUC/TRICOM vectorsDendritic cells used as APC rF-CEA/ Uninfected + Un- MUC/ Uninfected +MUC-1 T-cell line infected FP-WT TRICOM CEA peptide peptide T-rV <15.6<15.6 >1,000 976.3 514.0 T-rF <15.6 <15.6 >1,000 550.0 446.0 T-rF(CEA)<15.6 <15.6 403.9 729.2 <15.6 T-rF(MUC) <5.6 <15.6 381.4 <15.6 626.8Human T-cell lines T-rV, T-rF, T-rF(CEA) and T-rF(MUC) were generated asdescribed in the Materials and Methods section. These T-cell lines werestimulated with autologous uninfected DCs alone or pulsed with eitherthe CEA or MUC-1 peptide, DCs infected with the control vector FP-WT,and DCs infected with rF-CEA/IMUC/TRICOM. Peptides were used at aconcentration of 25 μg/ml; the effector-to-APC ratio was 10:1.Twenty-four hour culture supernatants were collected and screened forthe secretion of IFN-γ.

TABLE 14 Ability of T-cell lines established by using DCs infected withrV-CEA/MUC/TRICOM and rF-CEA/MUC/TRICOM as APC, to lyse human tumorcells C1R-A2 cells pulsed with No CEA MUC-1 PSA T-cell line Peptidepeptide′ peptide peptide MCF-7 SW1463 SK-Mel-24 T-rV −2.1 (1.26) 57.9(4.5)^(a) 50.0 (1.3)^(a) 0.6 (2.4) 34.4 (0.1)^(b) 27.8 (0.5)^(b) 1.6(1.1) T-rF 3.58 (3.9)  62.0 (4.6)^(a) 59.7 (0.8)^(a) 3.8 (0.2) 31.0(2.4)^(b) 25.6 (1.2)^(b) 2.7 (2.0) T-rF(CEA) 4.3 (1.6) 62.8 (1.9)^(a)−1.9 (1.0) 2.7 (1.0)  4.2 (3.2) 32.3 (2.1)^(b) 0.6 (3.5) T-rF(MUC) −1.6(3.2)  −3.6 (5.5) 46.4 (3.3)^(a) 1.4 (4.5) 38.2 (1.3)^(b) 25.2 (1.3)^(b)0.1 (1.1) Results are expressed in % lysis (SD). The human T-cell linesT-rV, T-rF, T-rF(CEA) and T-rF(MUC) were established as described in theMaterials and Methods section. A 6-hour ¹¹¹In release assay wasperformed on ClR-A2 cells and a 16-hour ¹¹¹In release assay wasperformed on MCF-7, SW1463 and SK-Mel-24 cells. CEA peptide (CAP1-6D),MUC-1 peptide (P-93L), and PSA peptide (PSA-3) were used at aconcentration of 25 μg/ml. MCF-7 (human breast carcinoma cell line:HLA-A2+, MUC-1-positive and CEA-negative); SW1463 (colon carcinoma cellline: HLA-A2+, MUC-1 positive and CEA positive); SK-Mel-24 (humanmelanoma cell line: HLA-A2+, MUC-1 negative, CEA negative). Theeffector-to-target ratio was 25:1. ^(a)Statistical significance (P <0.01, two-tailed t test) when comparing lysis to ClR-A2 cells.^(b)Statistical significance (P < 0.01, two-tailed t test) whencomparing lysis to SK-Mel-24 cells.

TABLE 15 Establishment of T-cell lines from cancer patients usingrF-CEA/MUC/TRICOM-infected autologous DCs demonstrates reactivity toboth CEA and MUC-1 epitopes Dendritic cells used as APC rF-CEA/MUC1/Uninfected + Uninfected + Uninfected + T-cell line Uninfected FP-WTTRICOM CEA peptide MUC-1 peptide PSA peptide T-55 <15.6<15.6 >1,000 >1,000 985.8 <15.6 T-49 <15.6 <15.6 >1,000 974.2 819.0<15.6 T-41 <15.6 <15.6 846.4 933.4 745.0 <15.6 T-55, T-49 and T-41 wereestablished by stimulating T cells isolated from a pancreatic cancerpatient (#55) and colon carcinoma patients (#49 and #41) with autologousDCs infected with rF-CEA/MUC/TRICOM (40 MOI), for three IVS. Theeffector-to-APC ratio was 10:1. Peptides were used at a concentration of25 μg/ml. Twenty-four hour culture supernatants were collected andscreened for the secretion of IFN-γ. Results are expressed in pg/ml ofIFN-γ.

TABLE 16 Ability of a T-cell line (T-55) established from a pancreaticpatient using rF- CEA/MUC/TRICOM-infected DCs as APC, to lyse humantumor cells C1R-A2 cells pulsed with No CEA MUC-1 PSA Effector:Targetpeptide Peptide^(a) peptide peptide MCF-7 SW1463 SK-Mel-24 50:1 0 (0.6)52.4 (3.3)^(a) 53.3 (0.4)^(a) 0 (0.1) 23.8 (1.2)^(b) 24.1 (1.2)^(b) 0.5(0.2) 25:1 0 (0.9) 30.3 (1.3)^(a) 34.4 (0.8)^(a) 0.6 (0.5)   20.4(1.1)^(b) 17.6 (0.3)^(b) 0.4 (0.3) 12.5:1   0 (0.2) 16.4 (1.8)^(a) 26.2(5.0)^(a) 0 (0.2) 13.8 (1.2)^(b) 15.2 (0.4)^(b)   0 (0.3) A 6-hour ¹¹¹Inrelease assay was performed on C1R-A2 cells and a 16-hour ¹¹¹In releaseassay was performed on MCF-7, SW1463 and SK-Mel-24 cells. Results areexpressed in % lysis (SD). CEA peptide (CAP1-6D), MUC-1 peptide (P-93L),and PSA peptide (PSA-3) were all used at a concentration of 25 μg/ml.MCF-7 (human breast carcinoma cell line: HLA-A2+, MUC-1-positive andCEA-negative); SW1463 (colon carcinoma cell line: HLA-A2+, MUC-1positive and CEA positive); SK-Mel-24 (human melanoma cell line:HLA-A2+, MUC-1 negative, CEA negative). T-55 was established bystimulating T cells isolated from a pancreatic patient (#55) withautologous DCs infected with rF-CEA/MUC/TRICOM (40 MOI) for three IVS.^(a)Statistical significance (P < 0.01, two-tailed t test) whencomparing lysis to C1R-A2 cells. ^(b)Statistical significance (P < 0.01,two-tailed t test) when comparing lysis to SK-Mel-24 cells.

TABLE 17 Ability of T-cell lines (T-49 and T-41) established from coloncarcinoma patients using rF-CEA/MUC/TRICOM-infected DCs as APC, to lysehuman tumor cells T-cell lines MCF-7 SW1463 SK-Mel-24 T-49 40:1 20.6(0.9)^(a) 42.9 (1.9)^(a) 0.8 (0.1) 20:1 12.0 (1.6)^(a) 30.2 (0.3)^(a)0.2 (0.2) 10:1  6.7 (1.0) 21.9 (2.1)^(a) 1.9 (0.8) T-41 40:1 24.4(1.6)^(a) 33.4 (1.3)^(a) 0.9 (0.3) 20:1 20.5 (1.2)^(a) 26.1 (1.1)^(a)1.2 (0.5) 10:1 11.0 (2.0)^(a) 20.1 (0.1)^(a) 2.0 (0.2) T-49 and T-41cell lines were established by stimulating T cells isolated from coloncarcinoma patients (#49 and #41) with autologous DCs infected withrF-CEA/MUC/TRICOM (40 MOI), for three IVS. A 16-hour ¹¹¹In release assaywas performed on MCF-7, SW1463 and SK-Mel-24 cells. Results areexpressed in % lysis (SD). MCF-7 (human breast carcinoma cell line:HLA-A2+, MUC-1-positive and CEA-negative); SW1463 (colon carcinoma cellline: HLA-A2+, MUC-1 positive and CEA positive); SK-Mel-24 (humanmelanoma cell line: HLA-A2+, MUC-1 negative, CEA negative).^(a)Statistical significance (P < 0.01, two-tailed t test) whencomparing lysis to SK-Mel-24 cells.

All documents mentioned herein are incorporated herein by reference.

The foregoing description is illustrative thereof, and it will beunderstood that variations and modifications can be effected withoutdeparting from the scope or spirit of the invention as set forth in thefollowing claims.

What is claimed is:
 1. An isolated polypeptide up to 12 amino acids inlength comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:7.
 2. The polypeptide of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:
 5. 3. The polypeptide of claim 1,wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
 4. An isolated nucleic acid molecule consisting of a nucleic acidsequence that encodes the polypeptide of claim
 1. 5. The nucleic acidmolecule of claim 4, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:
 5. 6. The nucleic acid molecule of claim 4,wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
 7. The nucleic acid molecule of claim 4, wherein the nucleic acidsequence comprises SEQ ID NO: 24 or SEQ ID NO:
 26. 8. A recombinantvector comprising a nucleic acid sequence encoding a polypeptide up to12 amino acids in length comprising the amino acid sequence of SEQ IDNO: 5 or SEQ ID NO:
 7. 9. The vector of claim 8, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:
 5. 10. The vector ofclaim 8, wherein the polypeptide comprises the amino acid sequence ofSEQ ID NO:
 7. 11. The vector of claim 8, wherein the nucleic acidsequence comprises SEQ ID NO: 24 or SEQ ID NO:
 26. 12. The vector ofclaim 8, wherein the nucleic acid sequence is operably linked to aninducible promoter.
 13. The vector of claim 8, wherein the vector is aviral vector or plasmid.
 14. A host cell comprising the vector of claim8.
 15. A method for treating a subject suffering from a MUC-1 tumorcomprising: isolating dendritic cells from a subject suffering fromcancer; transducing the dendritic cells with the recombinant vector ofclaim 8, and administering the transduced dendritic cells to thesubject, such that the subject is treated.
 16. A method for generatingan immune response to a MUC-1 tumor antigen in a subject comprisingadministering to the subject at least one nucleic acid molecule of claim4 in a therapeutically effective dose sufficient to generate a cellularimmune response in the subject.
 17. A method for treating a subjectsuffering from a MUC-1 tumor comprising administering to a subject atleast one polypeptide of claim 1, such that the subject is treated. 18.A method for treating a subject suffering from a MUC-1 tumor comprising:isolating dendritic cells from a subject suffering from cancer; treatingthe dendritic cells with at least one polypeptide of claim 1; and,administering the treated dendritic cells to the subject, such that thesubject is treated.
 19. A method for generating an immune response to aweakly immunogenic antigen comprising administering to a subject atleast one polypeptide of claim 1 fused to a weak immunogen.
 20. A methodfor treating a subject suffering from a MUC-1 tumor comprising:isolating dendritic cells from a subject suffering from cancer; treatingthe dendritic cells with at least one polypeptide of claim 1; activatingperipheral blood mononuclear cells with the treated dendritic cells;administering the activated PBMC cells to the subject.